Quinolinic acid phosphoribosyltransferase from Mycobacterium Tuberculosis (Mt-QAPRTase) is required for the de novo biosynthesis of NAD in both prokaryotes and eukaryotes (equivalent enzyme). The enzyme catalyses the reaction between quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP), to yield nicotinic acid mononucleotide (NAMN), pyrophosphate and CO2, the latter resulting from decarboxylation at position 2 of the quinolinate ring.
\r\n\r\nQAPRTase has been grouped with other phosphoribosyltransferases, (PRTases) that catalyse chemically similar phosphoribosyl transfer reactions using the substrate PRPP. The PRTases are involved in de novo and salvage reactions of nucleotide synthesis, as well as in histidine and tryptophan biosynthesis [PMID:9016724]. To date, crystal structures have been determined for several PRTase enzymes and all show a common 'PRTase fold' (the 'type I' fold) composed of a central beta sheet, of five beta strands, surrounded by alpha helices. The fold contains a common recognition motif of thirteen residues which is critical for PRPP binding and catalysis. However, as type II enzymes like Mt-QAPRTase lack the type I PRPP-binding motif and have TIM barrel-like structure, it becomes possible that there might be at least two different types of PRTase fold [PMID:9016724]. Despite their structural differences, it has been suggested TI and TII indeed still have the same catalytic mechanism but more work is needed to understand this fully.
","protein":{"sequences":[{"uniprot_id":"P9WJJ7"}]},"all_ecs":["2.4.2.19"],"residues":[{"mcsa_id":8,"roles_summary":"electrostatic stabiliser, proton acceptor, proton donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton donor","emo":"EMO_00068"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton acceptor","emo":"EMO_00066"}],"residue_chains":[{"chain_name":"A","pdb_id":"1qpr","assembly_chain_name":"A","assembly":1,"code":"Glu","resid":200,"auth_resid":201,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.20.20.70"}],"residue_sequences":[{"uniprot_id":"P9WJJ7","code":"Glu","is_reference":true,"resid":201}]},{"mcsa_id":8,"roles_summary":"electrostatic stabiliser, repulsive charge-charge interaction, steric role","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"","function_type":"interaction","function":"repulsive charge-charge interaction","emo":"EMO_00120"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"steric role","function_type":"spectator","function":"steric role","emo":"EMO_00029"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"A","pdb_id":"1qpr","assembly_chain_name":"A","assembly":1,"code":"Lys","resid":139,"auth_resid":140,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.20.20.70"}],"residue_sequences":[{"uniprot_id":"P9WJJ7","code":"Lys","is_reference":true,"resid":140}]},{"mcsa_id":8,"roles_summary":"electrostatic stabiliser","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"B","pdb_id":"1qpr","assembly_chain_name":"B","assembly":1,"code":"Arg","resid":104,"auth_resid":105,"is_reference":true,"domain_name":"B01","domain_cath_id":"3.90.1170.20"}],"residue_sequences":[{"uniprot_id":"P9WJJ7","code":"Arg","is_reference":true,"resid":105}]},{"mcsa_id":8,"roles_summary":"electrostatic stabiliser, proton acceptor, proton donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton acceptor","emo":"EMO_00066"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton donor","emo":"EMO_00068"}],"residue_chains":[{"chain_name":"A","pdb_id":"1qpr","assembly_chain_name":"A","assembly":1,"code":"Lys","resid":171,"auth_resid":172,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.20.20.70"}],"residue_sequences":[{"uniprot_id":"P9WJJ7","code":"Lys","is_reference":true,"resid":172}]},{"mcsa_id":8,"roles_summary":"electrostatic stabiliser","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"A","pdb_id":"1qpr","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":221,"auth_resid":222,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.20.20.70"}],"residue_sequences":[{"uniprot_id":"P9WJJ7","code":"Asp","is_reference":true,"resid":222}]}],"reaction":{"ec":"2.4.2.19","compounds":[{"count":1,"type":"reactant","chebi_id":"58017","name":"5-O-phosphonato-alpha-D-ribofuranosyl diphosphate(5-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/58017.mol"},{"count":1,"type":"reactant","chebi_id":"29959","name":"quinolinate(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/29959.mol"},{"count":1,"type":"product","chebi_id":"57502","name":"nicotinate D-ribonucleotide(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/57502.mol"},{"count":1,"type":"product","chebi_id":"16526","name":"carbon dioxide","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/16526.mol"},{"count":1,"type":"product","chebi_id":"33019","name":"diphosphate(3-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/33019.mol"},{"count":2,"type":"reactant","chebi_id":"15378","name":"hydron","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/15378_Fxpq4oZ.mol"}],"mechanisms":[{"mechanism_id":2,"is_detailed":true,"mechanism_text":"Phosphoribosyl transfer has been proposed to proceed via a unimolecular nucleophilic substitution (SN1 reaction) involving an oxycarbonium-like intermediate. In a rate-limiting step, the pyrophosphate group of PRPP is protonated and cleaved to yield an oxycarbonium of ribosylphosphate. The formation of the anticipated intermediate may be facilitated by the electron-withdrawing power of the metal ions and the C3-exo pucker of the ribosyl ring. Subsequently, the nucleophilic N1 of QA combines with the oxycarbonium in a diffusion-controlled reaction to form quinolinic acid mononucleotide (QAMN) [PMID:9862811].","rating":3,"components_summary":"proton transfer, overall product formed, bond polarisation, inferred reaction step, native state of enzyme is not regenerated, rate-determining step, unimolecular elimination by the conjugate base, intermediate formation, intermediate terminated, bimolecular nucleophilic addition, dephosphorylation, intermediate collapse, decarboxylation, overall reactant used, heterolysis","steps":[{"step_id":1,"description":"Glu201 deprotonates the 3-OH of the ribose substrate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_8_mechanism_2_step_1_r0n0xIc","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_8_mechanism_2_step_1_r0n0xIc.mrv"},{"step_id":2,"description":"The ribose intermediate undergoes an elimination reaction, with concomitant deprotonation of an unidentified base, represented here as a hydronium ion.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_8_mechanism_2_step_2_bVjB63g","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_8_mechanism_2_step_2_bVjB63g.mrv"},{"step_id":3,"description":"The nitrogen of the pyridine initiates a nucleophilic attack on the ribose in an addition reaction.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.8.2.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.8.2.3.mrv"},{"step_id":4,"description":"The pyridine intermediate decarboxylates with concomitant deprotonation of Lys172. Proton transfer to nicotinamide carbon is inferred.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.8.2.4","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.8.2.4.mrv"},{"step_id":5,"description":"The intermediate deprotonates Glu201.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.8.2.5","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.8.2.5.mrv"},{"step_id":6,"description":"Inferred return step. Lys172 returns to its positive state via a water molecule.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_8_2_6","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_8_2_6.mrv"},{"step_id":7,"description":"Products.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.8.2.7","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.8.2.7.mrv"}],"references":[{"pubmed_id":"9862811","doi":"10.1016/s0969-2126(98)00156-7","title":"Crystal structure of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis: a potential TB drug target","evidence_types":["required","crystallography evidence"]},{"pubmed_id":"9016724","doi":"10.1016/s0969-2126(97)00165-2","title":"A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase","evidence_types":["crystallography evidence","traceable author statement (general)"]},{"pubmed_id":"20047306","doi":"10.1021/bi9018225","title":"Roles for Cationic Residues at the Quinolinic Acid Binding Site of Quinolinate Phosphoribosyltransferase","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"18321072","doi":"10.1021/bi7020475","title":"Comprehensive X-ray Structural Studies of the Quinolinate Phosphoribosyl Transferase (BNA6) fromSaccharomyces cerevisiae‡","evidence_types":["crystallography evidence","multiple sequence alignment (conservation)"]}]}],"is_polymeric":false}},{"mcsa_id":9,"enzyme_name":"methionine adenosyltransferase","is_reference_uniprot_id":true,"reference_uniprot_id":"P31153","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/9/","description":"In biological systems, methyl groups are transferred from a small number of donors to a large number of acceptors. S-adenosylmethionine (AdoMet) is the most widespread of these donors, and is synthesised solely by the action of AdoMet synthase. \r\nGlycine N-methyltransferase (GNMT) isolated from Rattus norvegicus catalyses the transfer of a methyl group from S-adenosyl-methionine (SAM) to glycine to produce N-methylglycine (sarcosine) and S-adenosyl homocysteine (SAH). Unlike most SAM-dependent methyltransferases, GNMT is not strongly inhibited by SAH. This, coupled with no evidence of a physiological role for sarcosine, has led to the suggestion that GNMT plays a key role in modulating the SAM/SAH ratio in tissues where it is an important cofactor, and so controls methyltransferase activity.
\r\n\r\nGNMT exists as a dimer of dimers. Each subunit contains a molecular basket structure and a flexible N-terminal U-loop that can block the entrance to the basket of the partner subunit of the dimer. The U-loop competes with SAM for binding at the active site and so can regulate catalytic activity. GNMT binds first to SAM, which causes a conformational change, and then to glycine.
","protein":{"sequences":[{"uniprot_id":"P13255"}]},"all_ecs":["2.1.1.20"],"residues":[{"mcsa_id":23,"roles_summary":"activator","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"activator","function_type":"spectator","function":"activator","emo":"EMO_00038"}],"residue_chains":[{"chain_name":"A","pdb_id":"1xva","assembly_chain_name":"A","assembly":1,"code":"His","resid":142,"auth_resid":142,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.40.50.150"}],"residue_sequences":[{"uniprot_id":"P13255","code":"His","is_reference":true,"resid":143}]},{"mcsa_id":23,"roles_summary":"electrostatic stabiliser, hydrogen bond acceptor, hydrogen bond donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"A","pdb_id":"1xva","assembly_chain_name":"A","assembly":1,"code":"Tyr","resid":21,"auth_resid":21,"is_reference":true,"domain_name":"","domain_cath_id":""}],"residue_sequences":[{"uniprot_id":"P13255","code":"Tyr","is_reference":true,"resid":22}]},{"mcsa_id":23,"roles_summary":"electrostatic stabiliser, hydrogen bond acceptor, hydrogen bond donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"A","pdb_id":"1xva","assembly_chain_name":"A","assembly":1,"code":"Tyr","resid":194,"auth_resid":194,"is_reference":true,"domain_name":"A01","domain_cath_id":"3.30.46.10"}],"residue_sequences":[{"uniprot_id":"P13255","code":"Tyr","is_reference":true,"resid":195}]},{"mcsa_id":23,"roles_summary":"electrostatic stabiliser, hydrogen bond acceptor","function_location_abv":"main-C","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"A","pdb_id":"1xva","assembly_chain_name":"A","assembly":1,"code":"Gly","resid":137,"auth_resid":137,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.40.50.150"}],"residue_sequences":[{"uniprot_id":"P13255","code":"Gly","is_reference":true,"resid":138}]},{"mcsa_id":23,"roles_summary":"electrostatic stabiliser","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"}],"residue_chains":[{"chain_name":"A","pdb_id":"1xva","assembly_chain_name":"A","assembly":1,"code":"Arg","resid":175,"auth_resid":175,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.40.50.150"}],"residue_sequences":[{"uniprot_id":"P13255","code":"Arg","is_reference":true,"resid":176}]}],"reaction":{"ec":"2.1.1.20","compounds":[{"count":1,"type":"reactant","chebi_id":"59789","name":"S-adenosyl-L-methionine zwitterion","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/59789.mol"},{"count":1,"type":"product","chebi_id":"57856","name":"S-adenosyl-L-homocysteine zwitterion","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/57856.mol"},{"count":1,"type":"product","chebi_id":"15378","name":"hydron","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/15378_Fxpq4oZ.mol"},{"count":1,"type":"reactant","chebi_id":"57305","name":"glycine zwitterion","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/57305.mol"},{"count":1,"type":"product","chebi_id":"57433","name":"sarcosine zwitterion","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/57433.mol"}],"mechanisms":[{"mechanism_id":1,"is_detailed":true,"mechanism_text":"SAM and glycine bind in the active site in sequential order (SAM first). Glycine binds to the NE of tryptophan and the main chain carbonyl of alanine. Hydrogen bond interactions between glutamate and the SAM and glutamate and the glycine encourage the nucleophilic attack by glycine on the SAM methyl group. However, GNMT primarily catalyses the reaction through proximity and orientation effects. Arg175 favours the binding of the basic form of glycine (amine group neutral and carboxylate group deprotonated) over the zwitterionic form. Glycine and SAM are bound in such a way to align the lone pair orbital of the amino nitrogen of the amine and C-S bond of the methyl group of SAM. Tyr21 is thought to polarise this bond, though there is conflicting evidence on the subject. The thermal motion of the enzyme causes the two substrates to collide, leading to an Sn2 reaction to form sarcosine and SAH. The transition state of this reaction is stabilised by hydrogen bonds between the amine group of glycine and Gly137 and Tyr242. It is better stabilised when glycine is in the basic form than the zwitterionic form.","rating":3,"components_summary":"overall product formed, native state of cofactor is not regenerated, bimolecular nucleophilic substitution, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"The amine of the substrate glycine initiates a nucleophilic attack on the methyl of the S-adenosyl-L-methionine in a substitution reaction.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.23.1.1_4ieuE6z","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.23.1.1_4ieuE6z.mrv"},{"step_id":2,"description":"Products.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.23.1.2","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.23.1.2.mrv"}],"references":[{"pubmed_id":"8810903","doi":"10.1021/bi961068n","title":"Crystal Structure of GlycineN-Methyltransferase from Rat Liver†,‡","evidence_types":["crystallography evidence"]},{"pubmed_id":"17154529","doi":"10.1021/bi061319k","title":"Catalysis in GlycineN-Methyltransferase: Testing the Electrostatic Stabilization and Compression Hypothesis†","evidence_types":["biological system reconstruction (modelling)","crystallography evidence"]},{"pubmed_id":"12859184","doi":"10.1021/bi034245a","title":"Catalytic Mechanism of GlycineN-Methyltransferase†,∇","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"16851960","doi":"10.1021/jp0443254","title":"Methyl Transfer in GlycineN-Methyltransferase. A Theoretical Study","evidence_types":["computational experiment"]}]}],"is_polymeric":false}},{"mcsa_id":24,"enzyme_name":"4-chlorobenzoyl-CoA dehalogenase","is_reference_uniprot_id":true,"reference_uniprot_id":"A5JTM5","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/24/","description":"Chlorobenzoate dehalogenase catalyses the hydrolysis of chlorobenzoyl-CoA to hydroxybenzoyl-CoA. This reaction is used by bacteria as part of a three enzyme pathway for the utilisation of chlorinated organic compounds as a carbons source. The chlorobenzoate dehalogenase step is the second in the pathway and is structurally related to the crotonase-like superfamily of enzymes found in the beta-oxidation cycle. \r\nPhosphatidylinositol-specific phospholipase C (PI-PLC) is a ubiquitous enzyme involved in a vast range of cellular signalling cascades. Prokaryotic PLCs act as virulence factors in some bacteria, catalysing the hydrolysis of the sn-3 phosphodiester bond of phosphatidylinositol (PI) producing diacylglycerol (DAG) and inositol 1-phosphate (I(1)P).
\r\n\r\nFor the bacterial enzyme, the main product is the cyclic intermediate, myo-inositol 1,2-cyclic phosphate, which is rapidly hydrolysed to I(1)P by the eukaryotic PI-PLCs whereas the mammalian enzyme undergoes complete catalysis.
\r\n\r\nPI-PLC from Bacillus cereus, and the nearly identical enzyme from B. thuringiensis have been used as model systems for the study of IP-PLCs as a whole. Bacterial PI-PLCs are metal-ion-independent. This is in contrast to mammalian PI-PLCs which are Ca(II) dependent. The R69D mutant of B. thuringiensis PI-PLC is calcium dependent and has been used to investigate the role of calcium in catalysis by PI-PLCs [PMID:16042375].
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donor","emo":"EMO_00114"}],"residue_chains":[{"chain_name":"A","pdb_id":"1ptd","assembly_chain_name":"A","assembly":1,"code":"Arg","resid":69,"auth_resid":69,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.20.20.190"}],"residue_sequences":[{"uniprot_id":"P14262","code":"Arg","is_reference":true,"resid":100}]},{"mcsa_id":26,"roles_summary":"electrostatic stabiliser, hydrogen bond acceptor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic 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by the C2 hydroxyl of the substrate on the phosphorus producing IP and a myo-inositol 1,2-cyclic phosphate. This is catalysed by His32 and His82 acting as general base and general acid catalysts respectively.
\r\n\r\nThe second step is reverse of the first step but with water acting as the nucleophile. This results in the hydrolysis of a phosphorus-oxygen bond in myo-inositol 1,2-cyclic phosphate to give D-myo-inositol 1-phosphate. However, for the bacterial enzyme, this step is slow and the main product is myo-inositol 1,2-cyclic phosphate.
","rating":3,"components_summary":"proton transfer, overall product formed, inferred reaction step, rate-determining step, cyclisation, intramolecular nucleophilic substitution, proton relay, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"His32 deprotonates the hydroxy group adjacent to the phosphate, which initiates an intramolecular nucleophilic addition of the oxyanion to the phosphate, which proceeds through a pentavalent transition state, to eliminate the product alcohol in a substitution reaction, with concomitant deprotonation of His82.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_26_mechanism_1_step_1_SPdjed6","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_26_mechanism_1_step_1_SPdjed6.mrv"},{"step_id":2,"description":"His82 deprotonates water, which in turn deprotonates His32 in an inferred step.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.26.1.2_ygHWTJj","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.26.1.2_ygHWTJj.mrv"},{"step_id":3,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.26.1.3","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.26.1.3.mrv"}],"references":[{"pubmed_id":"11583175","doi":"10.1021/bi010958m","title":"A Catalytic Diad Involved in Substrate-Assisted Catalysis: NMR Study of Hydrogen Bonding and Dynamics at the Active Site of Phosphatidylinositol-Specific Phospholipase C†","evidence_types":["spectrometry evidence","traceable author statement (general)"]},{"pubmed_id":"9466937","doi":"10.1006/jmbi.1997.1490","title":"Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C","evidence_types":["biological system reconstruction (modelling)","crystallography evidence","multiple sequence alignment (conservation)","inferred from mutant phenotype"]},{"pubmed_id":"9521777","doi":"10.1021/bi972646i","title":"Mechanism of Phosphatidylinositol-Specific Phospholipase C: A Unified View of the Mechanism of Catalysis†,‡","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"16042375","doi":"10.1021/bi047896v","title":"X-ray Structure of the R69D Phosphatidylinositol-Specific Phospholipase C Enzyme: Insight into the Role of Calcium and Surrounding Amino Acids in Active Site Geometry and Catalysis","evidence_types":["crystallography evidence"]},{"pubmed_id":"9335537","doi":"10.1021/bi971102d","title":"Probing the Roles of Active Site Residues in Phosphatidylinositol-Specific Phospholipase C fromBacillus cereusby Site-Directed Mutagenesis†","evidence_types":["inferred from mutant phenotype","crystallography evidence","multiple sequence alignment (conservation)","direct assay evidence"]},{"pubmed_id":null,"doi":"10.1021/bi002371y","title":"Involvement of the Arg−Asp−His Catalytic Triad in Enzymatic Cleavage of the Phosphodiester Bond†","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"9048554","doi":"10.1021/bi962512p","title":"Structural Mapping of the Catalytic Mechanism for a Mammalian Phosphoinositide-Specific Phospholipase C†,‡","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"9367761","doi":"10.1006/jmbi.1997.1290","title":"Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes","evidence_types":["crystallography evidence"]},{"pubmed_id":"9838022","doi":"10.1016/s0005-2760(98)00125-8","title":"Families of phosphoinositide-specific phospholipase C: structure and function","evidence_types":["traceable author statement (general)"]}]}],"is_polymeric":false}},{"mcsa_id":27,"enzyme_name":"phospholipase C","is_reference_uniprot_id":true,"reference_uniprot_id":"P09598","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/27/","description":"Phospholipase C (PLC) from Bacillus cereus is a monomeric protein with a degree of similarly to mammalian PLCs and can mimic the action of mammalian PLCs for example by stimulating prostaglandin synthesis. \r\nPurple acid phosphatases (PAPs) are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters (e.g. of sugars or proteins) and anhydrides (e.g. ATP) under acidic conditions. The distinctive purple colour of these enzymes is due to a metal to ligand charge transfer from a tyrosine phenolate to a chromophoric Fe(III). The cornerstone of the active site of PAP is the presence of two metal ions; Fe(III) is always present in the chromophoric site, while the second site can be occupied by a redox active Fe(II/III) in mammals or a Zn(II) or Mn(II) in plants.
\r\n\r\nCrystal structures of human, pig, rat, and plant PAPs have been determined and show that the amino acid ligands of the metal ions are completely conserved across plant and animal PAPs, but there are some differences in the identities of the residues that line the active site.
","protein":{"sequences":[{"uniprot_id":"P80366"}]},"all_ecs":["3.1.3.2"],"residues":[{"mcsa_id":43,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"4kbp","assembly_chain_name":"A","assembly":1,"code":"His","resid":325,"auth_resid":325,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.60.21.10"}],"residue_sequences":[{"uniprot_id":"P80366","code":"His","is_reference":true,"resid":352}]},{"mcsa_id":43,"roles_summary":"electrostatic stabiliser, hydrogen bond 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ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"4kbp","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":135,"auth_resid":135,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.60.21.10"}],"residue_sequences":[{"uniprot_id":"P80366","code":"Asp","is_reference":true,"resid":162}]},{"mcsa_id":43,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal 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ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"4kbp","assembly_chain_name":"A","assembly":1,"code":"His","resid":286,"auth_resid":286,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.60.21.10"}],"residue_sequences":[{"uniprot_id":"P80366","code":"His","is_reference":true,"resid":313}]},{"mcsa_id":43,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal 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dianion","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/67140.mol"},{"count":1,"type":"product","chebi_id":"43474","name":"hydrogenphosphate","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/43474.mol"}],"mechanisms":[{"mechanism_id":1,"is_detailed":true,"mechanism_text":"On the basis of structural studies with bound phosphate and tungstate (inhibitor), and the observed inversion of the phosphorous configuration during catalysis, an SN2-type mechanism is proposed for PAP. The substrate phosphate is bound to the Me(II) ion via one of the non-esterified oxygen atoms and oriented by His202 and His296 (numbering for kidney bean enzyme) for in-line attack of an Fe(III)-bound hydroxide ion from a position opposite the esterified oxygen. These residues and the metal ions are presumed to stabilise the pentaco-ordinate transition state. Tentatively, His296 may then act as a general acid to protonate the leaving group, followed by the attack of a water molecule on the Fe(III) atom to release the inorganic phosphate.","rating":3,"components_summary":"proton transfer, decoordination from a metal ion, intermediate formation, coordination to a metal ion, bimolecular nucleophilic substitution, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"Metal activated water attacks the phosphate in a nucleophilic addition, resulting in a pentavalent transition state, which collapses to release the alcohol group, which gains a proton from His296.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_43_mechanism_1_step_1_62CreqU","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_43_mechanism_1_step_1_62CreqU.mrv"},{"step_id":2,"description":"The phosphate undergoes a rotation in the active site.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_43_mechanism_1_step_2_aKGCqH9","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_43_mechanism_1_step_2_aKGCqH9.mrv"},{"step_id":3,"description":"Water displaces one of the phosphate coordination bonds.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_43_mechanism_1_step_3_CTr5A5x","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_43_mechanism_1_step_3_CTr5A5x.mrv"},{"step_id":4,"description":"The metal coordinated water is now deprotonated by the leaving phosphate group, regenerating the active site. In the product release step, another water enters the active site, displacing the phosphate product.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_43_mechanism_1_step_4_aLt6WmN","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_43_mechanism_1_step_4_aLt6WmN.mrv"},{"step_id":5,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_43_mechanism_1_step_5_BsgcGEd","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_43_mechanism_1_step_5_BsgcGEd.mrv"}],"references":[{"pubmed_id":"8683579","doi":"10.1006/jmbi.1996.0354","title":"Mechanism of Fe(III) – Zn(II) Purple Acid Phosphatase Based on Crystal Structures","evidence_types":["required","crystallography evidence","multiple sequence alignment (conservation)"]},{"pubmed_id":"22943065","doi":"10.1111/cbdd.12001","title":"Identification of Purple Acid Phosphatase Inhibitors by Fragment-Based Screening: Promising New Leads for Osteoporosis Therapeutics","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"7770774","doi":null,"title":"Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site.","evidence_types":["crystallography evidence"]},{"pubmed_id":"22684363","doi":"10.1007/s12010-012-9694-8","title":"A Molecular Description of Acid Phosphatase","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"22698580","doi":"10.1021/ar300067g","title":"Binuclear Metallohydrolases: Complex Mechanistic Strategies for a Simple Chemical Reaction","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"15625111","doi":"10.1073/pnas.0407239102","title":"Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase","evidence_types":["crystallography evidence"]}]}],"is_polymeric":false}},{"mcsa_id":44,"enzyme_name":"alkaline phosphatase","is_reference_uniprot_id":true,"reference_uniprot_id":"P00634","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/44/","description":"Alkaline phosphatases are zinc and magnesium containing metalloenzymes which function optimally at high pH. They are found in all organisms except some plants, in the periplasmic space (E. coli), in vacuoles (fungi) or GPI-anchored to the external cell membrane (mammals). Alkaline phosphatase-type activity is involved in many cell processes, including metabolism of glycerolipids, folate, and xenobiotics. They cleave phosphomonoester bonds from various substrates, such as the end of a DNA molecule.","protein":{"sequences":[{"uniprot_id":"P00634"}]},"all_ecs":["3.1.3.1"],"residues":[{"mcsa_id":44,"roles_summary":"activator, electrostatic stabiliser, hydrogen bond donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"activator","function_type":"spectator","function":"activator","emo":"EMO_00038"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"electrostatic 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ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Glu","resid":322,"auth_resid":322,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Glu","is_reference":true,"resid":344}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":327,"auth_resid":327,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Asp","is_reference":true,"resid":349}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"His","resid":331,"auth_resid":331,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"His","is_reference":true,"resid":353}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":369,"auth_resid":369,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Asp","is_reference":true,"resid":391}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"His","resid":370,"auth_resid":370,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"His","is_reference":true,"resid":392}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"His","resid":412,"auth_resid":412,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"His","is_reference":true,"resid":434}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":153,"auth_resid":153,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Asp","is_reference":true,"resid":175}]},{"mcsa_id":44,"roles_summary":"hydrogen bond acceptor, hydrogen bond donor, metal ligand, nucleofuge, nucleophile, proton acceptor, proton donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton donor","emo":"EMO_00068"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton acceptor","emo":"EMO_00066"},{"group_function":"covalent catalysis","function_type":"reactant","function":"nucleophile","emo":"EMO_00054"},{"group_function":"covalent catalysis","function_type":"reactant","function":"nucleofuge","emo":"EMO_00058"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Ser","resid":102,"auth_resid":102,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Ser","is_reference":true,"resid":124}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Lys","resid":328,"auth_resid":328,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Lys","is_reference":true,"resid":350}]},{"mcsa_id":44,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"}],"residue_chains":[{"chain_name":"A","pdb_id":"1alk","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":51,"auth_resid":51,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.720.10"}],"residue_sequences":[{"uniprot_id":"P00634","code":"Asp","is_reference":true,"resid":73}]}],"reaction":{"ec":"3.1.3.1","compounds":[{"count":1,"type":"reactant","chebi_id":"15377","name":"water","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/15377_Sw86oy5.mol"},{"count":1,"type":"product","chebi_id":"30879","name":"alcohol","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/30879.mol"},{"count":1,"type":"reactant","chebi_id":"67140","name":"phosphate monoester dianion","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/67140.mol"},{"count":1,"type":"product","chebi_id":"43474","name":"hydrogenphosphate","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/43474.mol"}],"mechanisms":[{"mechanism_id":1,"is_detailed":true,"mechanism_text":"A metal-activated water molecule deprotonates the nucleophilic serine residue. Serine attacks the phosphomonoester in a substitution reaction which eliminates the alcohol. The alcohol anion deprotonates water, which initiates a nucleophilic attack on the covalently bound phosphorus in a substitution reaction which eliminates serine as an anion. Serine then deprotonates water to regenerate the starting state of the enzyme. \r\nThe enzyme has a novel mechanism: the carbanion generated by carbon dioxide loss is localised in an sp2 orbital perpendicular to the pi system of the pyrimidine. In all other decarboxylases, the carbanion is delocalised either into an adjacent carbonyl or into a covalently bound thiamin, pyridoxal or pyruvoyl cofactor.
\r\n\r\nThe mechanism is a bimolecular electrophilic substitution SE2 in which decarboxylation and protonation occur in a step-wise manner. Firstly, the anionic carboxylate of the substrate is positioned in a negatively charged region close to the carboxylate of Asp60 and Asp65(B) and the carbon of the pyrimidine destined to become the carbanium is close to the positive protonated Lys62. This allows destabilisation of the ground state and the stabilisation of negative charge accumulation in the transition state.
\r\n\r\nExtensive hydrogen bonding interactions within the active site provide the binding energy required to force the carboxylate groups into close proximity. As the reaction proceeds, the weakly basic C-C bond linking the carboxy group to the pyrimidine becomes progressively more basic until proton transfer from the adjacent Lys62 occurs.
","rating":3,"components_summary":"proton transfer, overall product formed, rate-determining step, unimolecular elimination by the conjugate base, decarboxylation, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"The substrate undergoes decarboxylation.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.1.1_jb79qHn","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.1.1_jb79qHn.mrv"},{"step_id":2,"description":"The substrate deprotonates Lys62 forming UMP.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.1.2_0uhqUDS","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.1.2_0uhqUDS.mrv"},{"step_id":3,"description":"Lys62 deprotonates water.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.1.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.1.3.mrv"},{"step_id":4,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.1.4","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.1.4.mrv"}],"references":[{"pubmed_id":"11114509","doi":"10.1016/s0959-440x(00)00148-2","title":"The structural basis for the remarkable catalytic proficiency of orotidine 5′-monophosphate decarboxylase","evidence_types":["multiple sequence alignment (conservation)","traceable author statement (general)"]},{"pubmed_id":"10757968","doi":"10.1021/bi992952r","title":"Structural Basis for the Catalytic Mechanism of a Proficient Enzyme: Orotidine 5‘-Monophosphate Decarboxylase†,‡","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"11900543","doi":"10.1021/bi015758p","title":"Mapping the Active Site−Ligand Interactions of Orotidine 5‘-Monophosphate Decarboxylase by Crystallography†,‡","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"17918849","doi":"10.1021/ja076222f","title":"Product Deuterium Isotope Effect for Orotidine 5‘-Monophosphate Decarboxylase: Evidence for the Existence of a Short-Lived Carbanion Intermediate","evidence_types":["direct assay evidence"]},{"pubmed_id":"12045113","doi":"10.1146/annurev.biochem.71.110601.135446","title":"Catalytic Proficiency: The Unusual Case of OMP Decarboxylase","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"18186641","doi":"10.1021/ja710384t","title":"Formation and Stability of a Vinyl Carbanion at the Active Site of Orotidine 5‘-Monophosphate Decarboxylase: pKaof the C-6 Proton of Enzyme-Bound UMP","evidence_types":["direct assay evidence"]},{"pubmed_id":"18839943","doi":"10.1021/ja801202j","title":"Mechanism of OMP Decarboxylation in Orotidine 5′-Monophosphate Decarboxylase","evidence_types":["computational experiment"]},{"pubmed_id":"21870810","doi":"10.1021/bi2012355","title":"Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Importance of Residues in the Orotate Binding Site","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"20441167","doi":"10.1021/ja102408k","title":"Product Deuterium Isotope Effects for Orotidine 5′-Monophosphate Decarboxylase: Effect of Changing Substrate and Enzyme Structure on the Partitioning of the Vinyl Carbanion Reaction Intermediate","evidence_types":["direct assay evidence"]},{"pubmed_id":"22620855","doi":"10.1021/bi300585e","title":"Orotidine 5′-Monophosphate Decarboxylase: Transition State Stabilization from Remote Protein–Phosphodianion Interactions","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"22812629","doi":"10.1021/ja3058474","title":"Proton Transfer from C-6 of Uridine 5′-Monophosphate Catalyzed by Orotidine 5′-Monophosphate Decarboxylase: Formation and Stability of a Vinyl Carbanion Intermediate and the Effect of a 5-Fluoro Substituent","evidence_types":["multiple sequence alignment (conservation)"]},{"pubmed_id":"23030629","doi":"10.1021/bi301188k","title":"Conformational Changes in Orotidine 5′-Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5′-Phosphate Group Activates the Enzyme","evidence_types":["direct assay evidence"]},{"pubmed_id":"24958125","doi":"10.1021/ja505037v","title":"Enzyme Architecture: Deconstruction of the Enzyme-Activating Phosphodianion Interactions of Orotidine 5′-Monophosphate Decarboxylase","evidence_types":["direct assay evidence"]},{"pubmed_id":"24559040","doi":"10.1080/07391102.2014.881303","title":"Study of orotidine 5′-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation","evidence_types":["computational experiment"]},{"pubmed_id":"20424759","doi":"10.1039/b926894d","title":"Interrogation of the active site of OMP decarboxylase from Escherichia coli with a substrate analogue bearing an anionic group at C6","evidence_types":["direct assay evidence"]},{"pubmed_id":"19039361","doi":"10.1039/b812979g","title":"A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: Mechanistic implications","evidence_types":["direct assay evidence"]},{"pubmed_id":"10681442","doi":"10.1073/pnas.259441296","title":"The crystal structure and mechanism of orotidine 5'-monophosphate decarboxylase","evidence_types":["inferred from mutant phenotype","crystallography evidence","multiple sequence alignment (conservation)"]},{"pubmed_id":"19472232","doi":"10.1002/chem.200900397","title":"Lys314 is a Nucleophile in Non-Classical Reactions of Orotidine-5′-Monophosphate Decarboxylase","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"19435314","doi":"10.1021/bi900623r","title":"Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Evidence for Substrate Destabilization,","evidence_types":["direct assay evidence"]}]},{"mechanism_id":3,"is_detailed":true,"mechanism_text":"This is the nucleophilic addition mechanism, which due to lack of evidence of deuterium isotope effect and failure to detect the addition of a nucleophile has been suggested against. There is also no evidence of what residues could be acting as the nucleophile. In this mechanism the decarboxylation is proceeded by the initial addition of a nucleophile to C5 of OMP and the formation of the vinyl carbanion is avoided by the concerted elimination of carbon dioxide and the active site nucleophile.","rating":1,"components_summary":"proton transfer, overall product formed, inferred reaction step, bimolecular nucleophilic addition, decarboxylation, overall reactant used, aromatic unimolecular elimination by the conjugate base, native state of enzyme regenerated","steps":[{"step_id":1,"description":"The enzyme initiates a nucleophilic attack on the substrate. The identity of the nucleophile is unknown and assumed to be Lys62 due to its proximity in the crystal structure.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.3.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.3.1.mrv"},{"step_id":2,"description":"Decarboxylation with concomitant elimination of the catalytic nucleophile.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.3.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.3.2.mrv"},{"step_id":3,"description":"Inferred return step to regenerate the protonation state of Lys33.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.3.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.3.3.mrv"},{"step_id":4,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.3.4","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.3.4.mrv"}],"references":[]},{"mechanism_id":2,"is_detailed":true,"mechanism_text":"This is the carbene mechanism, lack of deuterium isotope effect and failure to detect the addition of a nucleophile has been used to suggest that this mechanism is not favoured. There is also no evidence of what residues could be acting as the proton donor or nucleophile in the other mechanistic suggestions. In the carbene mechanism, protonation of the O4 carbonyl generates a carbocation at C6 of the substrate. Decarboxylation would then generate a stabilised carbene, rather than the high-energy vinyl carbanion","rating":1,"components_summary":"proton transfer, overall product formed, inferred reaction step, decarboxylation, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"Lys62 donates a proton to the activated sbstrate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.2.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.2.1.mrv"},{"step_id":2,"description":"Decarboxylation produces a stable carbene intermediate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.2.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.2.2.mrv"},{"step_id":3,"description":"Lys62 abstracts a proton from the intermediate to generate the final product.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.2.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.2.3.mrv"},{"step_id":4,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.50.2.4","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.50.2.4.mrv"}],"references":[]}],"is_polymeric":false}},{"mcsa_id":51,"enzyme_name":"phosphoenolpyruvate carboxykinase (ATP)","is_reference_uniprot_id":true,"reference_uniprot_id":"P22259","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/51/","description":"Phosphoenolpyruvate carboxykinase (PEPCK) catalyses first committed (rate-limiting) step in hepatic gluconeogenesis, namely the reversible decarboxylation of oxaloacetate to phosphoenolpyruvate (PEP) and carbon dioxide. This reaction can occur using either ATP or GTP as a source of the phosphate. The ATP-utilising (EC:4.1.1.49, represented here) and GTP-utilising (EC:4.1.1.32) enzymes form two divergent subfamilies, which have little sequence similarity but which retain conserved active site residues.\r\nThe allosteric hexameric enzyme from Escherichia coli catalyses the regulatory step of N-acetyl glucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia.The enzyme is a hexamer of identical subunits arranged as a dimer of trimers and the allosteric sites appear located in the clefts between the subunits forming the trimers.
\r\n\r\nGlucosamine-6-phosphate isomerase is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination.
\r\n\r\nThe allosteric transition from T to R is generated upon binding of GlcNAc6P at the allosteric site or binding of active-site ligands (GlcN6P, Fru6P, GlcN-ol-6P). An important local conformational change relating allosteric control to catalysis is centred on residue Glu148. Glu148 participates in a proton-relay system that serves to polarise His143. This histidine is essential for the catalytic ring opening of the GlcN6P substrate. The His143 is primarily activated by its interaction with Glu148. The second residue in the His-Glu-Asx triangle appears to be either an Asp or Asn. Interestingly enough, in the work on Escherichia coli, Asn in this position severely disrupts the enzyme's activity.
","protein":{"sequences":[{"uniprot_id":"P0A759"}]},"all_ecs":["3.5.99.6"],"residues":[{"mcsa_id":60,"roles_summary":"activator, hydrogen bond acceptor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"activator","function_type":"spectator","function":"activator","emo":"EMO_00038"},{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond acceptor","emo":"EMO_00113"},{"group_function":"","function_type":"interaction","function":"hydrogen bond 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6-phosphate(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/57634.mol"},{"count":1,"type":"reactant","chebi_id":"75989","name":"alpha-D-glucosamine 6-phosphate(1-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/75989.mol"},{"count":1,"type":"product","chebi_id":"28938","name":"ammonium","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/28938.mol"}],"mechanisms":[{"mechanism_id":1,"is_detailed":true,"mechanism_text":"Once the ring has been opened, Asp72 deprotonates the carbon to which the amine group is attached in an assisted keto-enol tautomerisation reaction. Then water is added in an electrophlic addition across the C1-C2 pi bond. Ammonia is then eliminated and the final product of the active site is the linear product, which undergoes spontaneous cyclisation.","rating":3,"components_summary":"bimolecular elimination, decyclisation, proton transfer, overall product formed, reaction occurs outside the enzyme, deamination, intermediate formation, intermediate terminated, cyclisation, assisted keto-enol tautomerisation, bimolecular electrophilic addition, intermediate collapse, proton relay, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"In a ring opening reaction, His143 deprotonates the C1 hydroxide.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.1_AIVOC2A","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.1_AIVOC2A.mrv"},{"step_id":2,"description":"The oxyanion collapses, cleaving the C-O bond with concomitant deprotonation of His143 by the newly formed oxyanion.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.2_hGheTAU","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.2_hGheTAU.mrv"},{"step_id":3,"description":"Asp72 deprotonates the carbon to which the amine group is attached.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.3_0qgRSJi","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.3_0qgRSJi.mrv"},{"step_id":4,"description":"The newly formed carbanion initiates a double bond rearrangement and deprotonates the Asp72.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.4_QKrQowM","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.4_QKrQowM.mrv"},{"step_id":5,"description":"In an electrophilic reaction, water is added across the C1-C2 pi bond. The water molecule approaches the re-face of the intermediate (i.e. from the same side as Asp72).","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.5_x9dXKK6","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.5_x9dXKK6.mrv"},{"step_id":6,"description":"Asp72 deprotonates the newly added hydroxyl group, eliminating ammonia from the substrate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.6_ibdnfGz","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.6_ibdnfGz.mrv"},{"step_id":7,"description":"The released ammonia deprotonates Asp72 to form ammonium and regenerate the enzyme starting state.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.1.7_ISct5uC","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.1.7_ISct5uC.mrv"},{"step_id":8,"description":"The linear product undergoes spontaneous cyclisation outside of the enzyme active site.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_60_1_8","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_60_1_8.mrv"},{"step_id":9,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_60_1_9","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_60_1_9.mrv"}],"references":[{"pubmed_id":"8747459","doi":"10.1016/s0969-2126(01)00270-2","title":"Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution","evidence_types":["crystallography evidence"]},{"pubmed_id":"11513596","doi":"10.1021/bi0105835","title":"On the Multiple Functional Roles of the Active Site Histidine in Catalysis and Allosteric Regulation ofEscherichia coliGlucosamine 6-Phosphate Deaminase†","evidence_types":["inferred from mutant phenotype","multiple sequence alignment (conservation)"]},{"pubmed_id":"1734962","doi":null,"title":"Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesis.","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"18436239","doi":"10.1016/j.jmb.2008.03.031","title":"Ring-Opening Mechanism Revealed by Crystal Structures of NagB and Its ES Intermediate Complex","evidence_types":["crystallography evidence","match to InterPro member signature (homology)"]},{"pubmed_id":"25069951","doi":"10.1039/c4cp01609b","title":"A QM/MM MD study of the pH-dependent ring-opening catalysis and lid motif flexibility in glucosamine 6-phosphate deaminase","evidence_types":["computational experiment"]},{"pubmed_id":"16168949","doi":"10.1016/j.abb.2005.08.002","title":"On the functional role of Arg172 in substrate binding and allosteric transition in Escherichia coli glucosamine-6-phosphate deaminase","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"15755726","doi":"10.1074/jbc.m502131200","title":"Structure and Kinetics of a Monomeric Glucosamine 6-Phosphate Deaminase: MISSING LINK OF THE NagB SUPERFAMILY?","evidence_types":["multiple sequence alignment (conservation)","traceable author statement (general)"]},{"pubmed_id":"10378272","doi":"10.1016/s0969-2126(99)80069-0","title":"The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 Å resolution","evidence_types":["traceable author statement (general)"]}]},{"mechanism_id":2,"is_detailed":true,"mechanism_text":"Once the ring has been opened, Asp72 deprotonates the carbon to which the amine group is attached in an assisted keto-enol tautomerisation reaction. Then a cis-enolamine is formed by the deprotonation of the amine group, water then adds to the the activated intermediate. The unstable carbinol ammonium intermediate then eliminates ammonia and the final product of the active site is the linear product, which undergoes spontaneous cyclisation.","rating":2,"components_summary":"bimolecular elimination, decyclisation, proton transfer, overall product formed, reaction occurs outside the enzyme, assisted tautomerisation (not keto-enol), intermediate formation, intermediate terminated, bimolecular nucleophilic addition, cyclisation, assisted keto-enol tautomerisation, bimolecular electrophilic addition, proton relay, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"In a ring opening reaction, His143 deprotonates the C1 hydroxide.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.1.mrv"},{"step_id":2,"description":"The oxyanion collapses, cleaving the C-O bond with concomitant deprotonation of His143 by the newly formed oxyanion.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.2.mrv"},{"step_id":3,"description":"Asp72 deprotonates the carbon to which the amine group is attached.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.3.mrv"},{"step_id":4,"description":"The newly formed carbanion initiates a double bond rearrangement and deprotonates the Asp72.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.4","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.4.mrv"},{"step_id":5,"description":"The proton from the ammonium group is transferred to the Asp72 carboxylate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.5","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.5.mrv"},{"step_id":6,"description":"cis-enolamine then removes the proton from Asp72 to the position C1 pro-R of the intermediate which rearranges to form fructosimine 6-phosphate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.6","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.6.mrv"},{"step_id":7,"description":"The imino bond reacts with a water molecule, giving an unstable carbinol ammonium intermediate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_60_2_7","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_60_2_7.mrv"},{"step_id":8,"description":"Asp72 deprotonates the newly added hydroxyl group, eliminating ammonia from the substrate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_60_2_8","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_60_2_8.mrv"},{"step_id":9,"description":"The released ammonia deprotonates Asp72 to form ammonium and regenerate the enzyme starting state.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.60.2.9","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.60.2.9.mrv"},{"step_id":10,"description":"The linear product undergoes spontaneous cyclisation outside of the enzyme active site.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_60_2_10","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_60_2_10.mrv"},{"step_id":11,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_60_2_11","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_60_2_11.mrv"}],"references":[{"pubmed_id":null,"doi":"10.1016/S0969-2126(01)00270-2","title":"Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 å resolution","evidence_types":["crystallography evidence"]}]}],"is_polymeric":false}},{"mcsa_id":61,"enzyme_name":"2-hydroxymuconate tautomerase","is_reference_uniprot_id":true,"reference_uniprot_id":"Q01468","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/61/","description":"2-hydroxymuconate tautomerase is also known as 4-oxalocrotonate tautomerase (4-OT). Converts 2-hydroxymuconate to the alpha-beta-unsaturated ketone, 2-oxo-3-hexenedioate [PMID:1339435]. This enzyme forms part of a bacterial metabolic pathway that oxidatively catabolises toluene, o-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene into intermediates of the citric acid cycle. \r\nUracil-DNA glycosylase (UDG) are monofunctional glycosylases and initiate the base excision repair (BER) pathway for uracil by hydrolysing the N-C'1 glycosylic bond between a target uracil and an abasic site. The human BER cycle is important for restoring the chemical integrity of DNA.
\r\n\r\nThe uracil-DNA glycosylase superfamily consists of 6 smaller families, based on sequence alignments. Both human and E.coli UDG come under Family I and are also called UNGs.
\r\n\r\nThe enzyme mechanism has been hotly debated. Classically, an acid/base mechanism has been employed (mechanism proposal 2), but new evidence suggests a steric distortion of the DNA substrate catalyses the reaction in a mechanism similar to SN1 dissociation (mechanism proposal 1).
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donor","emo":"EMO_00114"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"","function_type":"interaction","function":"covalently attached","emo":"EMO_00115"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton acceptor","emo":"EMO_00066"},{"group_function":"covalent catalysis","function_type":"reactant","function":"nucleophile","emo":"EMO_00054"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton donor","emo":"EMO_00068"},{"group_function":"covalent catalysis","function_type":"reactant","function":"nucleofuge","emo":"EMO_00058"}],"residue_chains":[{"chain_name":"A","pdb_id":"1eug","assembly_chain_name":"A","assembly":1,"code":"His","resid":187,"auth_resid":187,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.470.10"}],"residue_sequences":[{"uniprot_id":"P12295","code":"His","is_reference":true,"resid":187}]},{"mcsa_id":71,"roles_summary":"activator, electrostatic stabiliser, increase acidity, proton acceptor, proton donor","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"activator","function_type":"spectator","function":"activator","emo":"EMO_00038"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"activator","function_type":"spectator","function":"activator","emo":"EMO_00038"},{"group_function":"activator","function_type":"spectator","function":"increase acidity","emo":"EMO_00041"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"activator","function_type":"spectator","function":"increase acidity","emo":"EMO_00041"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton acceptor","emo":"EMO_00066"},{"group_function":"proton shuttle (general acid/base)","function_type":"reactant","function":"proton donor","emo":"EMO_00068"}],"residue_chains":[{"chain_name":"A","pdb_id":"1eug","assembly_chain_name":"A","assembly":1,"code":"Asp","resid":64,"auth_resid":64,"is_reference":true,"domain_name":"A00","domain_cath_id":"3.40.470.10"}],"residue_sequences":[{"uniprot_id":"P12295","code":"Asp","is_reference":true,"resid":64}]}],"reaction":{"ec":"3.2.2.3","compounds":[{"count":1,"type":"reactant","chebi_id":"15377","name":"water","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/15377_Sw86oy5.mol"},{"count":1,"type":"product","chebi_id":"17568","name":"uracil","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/17568.mol"},{"count":1,"type":"product","chebi_id":"90761","name":"2-deoxy-D-ribofuranose","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/90761.mol"},{"count":1,"type":"reactant","chebi_id":"16450","name":"2'-deoxyuridine","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/16450.mol"}],"mechanisms":[{"mechanism_id":1,"is_detailed":true,"mechanism_text":"This mechanism represents the steric contortion mechanism. Here the tetrahedral distortion is imposed by the structurally rigid walls of the active site, formed by Tyr and Phe residues. The enzyme centre flattens the pucker ring of the uridine deoxyribose, raising the glycosylic bond to a semi-axial position, allowing pi-sigma* overlap. This stereoelectronic effect increases in strength as the substrate is further distorted towards the transition state. As the transition state move towards a tetrahedral geometry, and glycosylic bond rotation occurs, an anomeric effect is coupled to the pi systems of the uracil ring, resulting in orbital overlap of the glycosylic bond and the carbonyl C2 and C4 pi systems. The developing negative charge of the transition state is stabilised by hydrogen bonding to His-187. The enzyme funnels binding energy for use is catalysis by employing substrate distortions to couple two stereoelectronic effects to promote efficient catalysis. The abasic nucleotide relaxes into a more puckered conformation and withdraws from the enzyme while uracil tilts deeper into the active site, improving its stacking interactions with the Phe residue present. These rearrangements lower the product's energies relative to the reactant's, reducing the strain within the active site and allowing the enzyme to bind preferentially to the products.","rating":3,"components_summary":"reaction occurs outside the enzyme, proton transfer, overall product formed, intermediate formation, intermediate terminated, bimolecular nucleophilic addition, intramolecular elimination, tautomerisation (not keto-enol), overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"The structural constraints imposed upon the uridine deoxyribose ring by Tyr 66 and Phe 77 induce unimolecular elimination to form a transient oxonium species and an anionic precursor of uracil","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.1.1_gNCm91I","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.1.1_gNCm91I.mrv"},{"step_id":2,"description":"The transient oxonium species is attacked by water, forming the 1-alpha hydroxy group of the D-ribose product. The anionic intermediate is stabilised though hydrogen bonding to His187, and is then protonated from the attacking water.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.1.2_3yvXSPL","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.1.2_3yvXSPL.mrv"},{"step_id":3,"description":"The intermediate tautomerises to form the uracil product.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.1.3_IEB7WpN","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.1.3_IEB7WpN.mrv"},{"step_id":4,"description":"Products.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.1.4","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.1.4.mrv"}],"references":[{"pubmed_id":"10946228","doi":"10.1016/s0921-8777(00)00026-4","title":"Lessons learned from structural results on uracil-DNA glycosylase","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"10090282","doi":"10.2210/pdb1eug/pdb","title":"Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited.","evidence_types":["pH","crystallography evidence","inferred from mutant phenotype"]},{"pubmed_id":"11716455","doi":"10.1006/abbi.2001.2605","title":"Uracil DNA Glycosylase: Insights from a Master Catalyst","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"7697717","doi":null,"title":"Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.","evidence_types":["inferred from mutant phenotype","crystallography evidence","traceable author statement (general)","direct assay evidence"]},{"pubmed_id":"7845459","doi":"10.1038/373487a0","title":"The structural basis of specific base-excision repair by uracil–DNA glycosylase","evidence_types":["crystallography evidence","multiple sequence alignment (conservation)"]},{"pubmed_id":"25252105","doi":"10.1002/pro.2554","title":"Uracil-DNA glycosylases-Structural and functional perspectives on an essential family of DNA repair enzymes","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"25181003","doi":"10.1039/c4ob01063a","title":"Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA.","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"11607036","doi":"10.1038/35099587","title":"Uracil-DNA glycosylase acts by substrate autocatalysis.","evidence_types":["computational experiment"]},{"pubmed_id":"11859082","doi":"10.1074/jbc.m200634200","title":"Probing the Limits of Electrostatic Catalysis by Uracil DNA Glycosylase Using Transition State Mimicry and Mutagenesis","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"12590578","doi":"10.1021/bi027014x","title":"Powering DNA Repair through Substrate Electrostatic Interactions†","evidence_types":["chemical modification"]},{"pubmed_id":"10805771","doi":"10.1073/pnas.97.10.5083","title":"Uracil-DNA glycosylase-DNA substrate and product structures: Conformational strain promotes catalytic efficiency by coupled stereoelectronic effects","evidence_types":["biological system reconstruction (modelling)","crystallography evidence"]}]},{"mechanism_id":2,"is_detailed":true,"mechanism_text":"This proposal represents the classical acid/base proposal carried out by a cationic His-187 and an absolutely conserved Asp-64. Here the peptide carbonyl and side-chain carboxyl of Asp-64 activate a water molecule that attacks a weakened glycosylic bond. Destabilisation of the N1−C1‘ bond is brought about by distortion or protonation of the uracil O2 by His-187 Nε2.","rating":2,"components_summary":"proton transfer, overall product formed, assisted tautomerisation (not keto-enol), intermediate formation, bimolecular nucleophilic substitution, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"Asp64 abstracts a proton from the catalytic water molecule. The activated hydroxide then attacks the anomeric carbon in a nucleophilic substitution reaction.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.2.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.2.1.mrv"},{"step_id":2,"description":"The intermediate abstracts a proton from Asp64 to regenerate the active site and form the final product.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.2.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.2.2.mrv"},{"step_id":3,"description":"Products.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.2.3","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.2.3.mrv"}],"references":[{"pubmed_id":"10200172","doi":"10.1021/bi982986j","title":"Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis.","evidence_types":["inferred from mutant phenotype"]}]},{"mechanism_id":3,"is_detailed":true,"mechanism_text":"The imidazole group of His-187 catalyses a direct nucleophilic attack on the N1−C1‘ glycosylic bond of uracil and a second nucleophilic attack of a water molecule provides H- and OH-group addition to the N1 and C1‘ atoms, respectively.","rating":1,"components_summary":"proton transfer, overall product formed, enzyme-substrate complex cleavage, intermediate formation, bimolecular nucleophilic substitution, overall reactant used, native state of enzyme regenerated, enzyme-substrate complex formation","steps":[{"step_id":1,"description":"His187 initiates a nucleophilic attack on the anomeric carbon of the DNA base.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.3.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.3.1.mrv"},{"step_id":2,"description":"The uracil undergoes tautomerisation and abstracts a proton from the catalytic water molecule, forming one of the final products and activating the water.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.3.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.3.2.mrv"},{"step_id":3,"description":"The activated water then attacks the anomeric carbon of the enzyme bound sugar. This forms the final product and regenerates the active site.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.3.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.3.3.mrv"},{"step_id":4,"description":"Products.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.3.4","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.3.4.mrv"}],"references":[{"pubmed_id":"7697717","doi":null,"title":"Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.","evidence_types":["inferred from mutant phenotype","crystallography evidence"]}]},{"mechanism_id":4,"is_detailed":true,"mechanism_text":"This proposal represents the second acid/base proposal carried out by a neutral His-187 and an absolutely conserved Asp-64. Here His-187 activates a water molecule that attacks a weakened glycosylic bond.","rating":1,"components_summary":"proton transfer, overall product formed, assisted tautomerisation (not keto-enol), intermediate formation, bimolecular nucleophilic substitution, overall reactant used, native state of enzyme regenerated","steps":[{"step_id":1,"description":"His-187 abstracts a proton from the catalytic water molecule. The activated hydroxide then attacks the anomeric carbon in a nucleophilic substitution reaction.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.4.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.4.1.mrv"},{"step_id":2,"description":"The intermediate abstracts a proton from His-187 to regenerate the active site and form the final product.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.4.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.4.2.mrv"},{"step_id":3,"description":"Products.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.71.4.3","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.71.4.3.mrv"}],"references":[{"pubmed_id":"25181003","doi":"10.1039/c4ob01063a","title":"Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA.","evidence_types":["traceable author statement (general)"]}]}],"is_polymeric":true}},{"mcsa_id":72,"enzyme_name":"L-fuculose-phosphate aldolase","is_reference_uniprot_id":true,"reference_uniprot_id":"P0AB87","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/72/","description":"L-fuculose-phosphate aldolase (FucA) catalyses the cleavage of L-fuculose-1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde , a conversion from a six carbon unit to a three carbon unit. FucA is a homotetramer with 215 amino acid residues and one zinc ion per subunit. \r\nPLA2 catalyses the hydroylsis of the 2-acyl ester of 1,2-diacylphosphatides. Two forms of PLA2 are known: the small secretory PLA2s which are amphipathic molecules usually found associated with lipid membranes, and the larger cytosolic forms. This annotation is for the small secretory form. Secretory PLA2s form part of the neurotoxic component of many snake and bee venoms due to its ability to block acetylcholine release.
\r\n\r\nSecretory PLA2s have been divided into 3 main groups: I, II, and III. Class I (present in some snake venoms and in mammalian exocrine pancrease) and II (present in some snake venoms and also broadly distributed among a variety of mammalian cell types) are closely related to each other. The class III enzymes (including venom enzymes from the honeybee and the Gila monster) appear to form a separate divergent group though their active site and mechanism is similar. The key catalytic histidine and aspartate are in very similar orientations however the residues making up the rest of the hydrogen bond network are not conserved spatially.
","protein":{"sequences":[{"uniprot_id":"P00592"}]},"all_ecs":["3.1.1.4"],"residues":[{"mcsa_id":83,"roles_summary":"electrostatic stabiliser, hydrogen bond donor","function_location_abv":"main-N","main_annotation":"","ptm":"","roles":[{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"","function_type":"interaction","function":"hydrogen bond donor","emo":"EMO_00114"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic stabiliser","emo":"EMO_00033"},{"group_function":"electrostatic interaction","function_type":"spectator","function":"electrostatic 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Ureases have been isolated from a wide range of bacteria, fungi and higher plants where it allows the organism to use urea as a nitrogen source. Ureases uses an almost unique bi-nickel catalytic centre which is liganded by a carbamylated lysine.
\r\n\r\nThe mechanism of this enzyme has been subject to debate since the early 1920s and the precise steps in catalysis remain unclear [PMID:20471401].
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Carbamylated lysine 217 provides an oxygen ligand to each nickel. One nickel ion is coordinated by three ligands: two histidines (246 and 272) and the carbamylated lysine 217 (with low occupancy of a fourth ligand) and the second is coordinated by five ligands: two histidines (136 and 134), aspartate 360, a water and the carbamylated lysine 217. Urea enters the active site and ligates to Ni-1 to complete its tetrahedral coordination. It also forms a hydrogen bond to histidine 219 which polarises the urea carbonyl. Histidine 320 acts as a general base and abstracts a proton from the Ni-2 water ligand, the resulting hydroxide ligand of Ni-2 then attacks the urea carbonyl carbon. The resulting tetrahedral intermediate decomposes, with the assistance of an unidentified general acid, to ammonia and Ni bound carbamylate which then dissociates.","rating":3,"components_summary":"reaction occurs outside the enzyme, proton transfer, unimolecular elimination by the conjugate base, deamination, bimolecular nucleophilic addition, intramolecular elimination","steps":[{"step_id":1,"description":"Asp221 deprotonates His219, which in turn deprotonates the nickel bound urea, forming the active conformer for this mechanism.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.2.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.2.1.mrv"},{"step_id":2,"description":"His320 deprotonates nickel bound water, which initiates a nucleophilic attack on the nickel bound urea in an addition reaction.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.2.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.2.2.mrv"},{"step_id":3,"description":"The tetrahedral intermediate collapses, eliminating ammonia with concomitant deprotonation of His320.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.2.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.2.3.mrv"},{"step_id":4,"description":"Carbamate dissociates from the active site and is spontaneously converted to carbon dioxide and a second molecule of ammonia","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_87_2_4","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_87_2_4.mrv"},{"step_id":5,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.2.5","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.2.5.mrv"}],"references":[{"pubmed_id":"22670767","doi":"10.1021/ja3043239","title":"Wide-Open Flaps Are Key to Urease Activity","evidence_types":["crystallography evidence"]},{"pubmed_id":"20886006","doi":"10.1155/2010/364891","title":"Computational Modeling of the Mechanism of Urease","evidence_types":["computational experiment"]},{"pubmed_id":null,"doi":"10.1016/j.molcatb.2009.01.003","title":"Ureases I. Functional, catalytic and kinetic properties: A review","evidence_types":["traceable author statement (general)"]},{"pubmed_id":null,"doi":"10.1021/ja000202v","title":"Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel Center: An Alternative Model for the Mechanism of Urease","evidence_types":["crystallography evidence"]},{"pubmed_id":"8702515","doi":"10.1074/jbc.271.31.18632","title":"Characterization of the Mononickel Metallocenter in H134A Mutant Urease","evidence_types":["required","inferred from mutant phenotype"]},{"pubmed_id":"8718850","doi":"10.1021/bi960424z","title":"Structures of theKlebsiella aerogenesUrease Apoenzyme and Two Active-Site Mutants†,‡","evidence_types":["required","crystallography evidence"]},{"pubmed_id":"8318888","doi":"10.1002/pro.5560020616","title":"Site-directed mutagenesis ofKlebsiella aerogenesurease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis","evidence_types":["inferred from mutant phenotype","multiple sequence alignment (conservation)"]},{"pubmed_id":"10913264","doi":"10.1021/bi000613o","title":"Kinetic and Structural Characterization of Urease Active Site Variants†,‡","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"9558361","doi":"10.1021/bi980021u","title":"Chemical Rescue ofKlebsiella aerogenesUrease Variants Lacking the Carbamylated-Lysine Nickel Ligand†,‡","evidence_types":["inferred from mutant phenotype"]},{"pubmed_id":"7754395","doi":"10.1126/science.7754395","title":"The crystal structure of urease from Klebsiella aerogenes","evidence_types":["crystallography evidence"]}]},{"mechanism_id":4,"is_detailed":true,"mechanism_text":"A third proposal is the elimination reaction from Barios and Lippard [PMID:11300826] has also been proposed on the observation of cyanic intermediates. Theoretical work by Estiu and Merz [PMID:16584179, PMID:17676790] suggests that the elimination pathway may occur in competition with the more traditionally proposed mechanisms.","rating":3,"components_summary":"reaction occurs outside the enzyme, inferred reaction step, bimolecular nucleophilic addition, intramolecular elimination, native state of enzyme regenerated","steps":[{"step_id":1,"description":"Asp221 deprotonates His219, which in turn abstracts a proton from the Ni(II) bound urea, forming a negatively charged intermediate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.4.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.4.1.mrv"},{"step_id":2,"description":"His320 deprotonats Asp221.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.4.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.4.2.mrv"},{"step_id":3,"description":"The negatively charged intermediate collapses, eliminating ammoinia, which abstracts the proton from His320.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.4.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.4.3.mrv"},{"step_id":4,"description":"Cyanic acid is the product of this enzyme mechanism, thus it is likely that the final hydrolysis occurs outside of the active site.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_87_4_4","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_87_4_4.mrv"},{"step_id":5,"description":"Ammonia is eliminated in the final non-enzyme catalysed step of the reaction.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_87_4_5","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_87_4_5.mrv"},{"step_id":6,"description":"Inferred step to regenerate the enzyme's active site. Two water molecules displace the product to form the ground state. It is unclear how the His219 returns to it's native state, we have represented a water facilitated tautomerisation reaction here.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/macie_entry_87_4_6","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/macie_entry_87_4_6.mrv"},{"step_id":7,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.4.7","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.4.7.mrv"}],"references":[{"pubmed_id":"8702515","doi":"10.1074/jbc.271.31.18632","title":"Characterization of the Mononickel Metallocenter in H134A Mutant Urease","evidence_types":["required","inferred from mutant phenotype"]},{"pubmed_id":"22670767","doi":"10.1021/ja3043239","title":"Wide-Open Flaps Are Key to Urease Activity","evidence_types":["crystallography evidence"]},{"pubmed_id":null,"doi":"10.1016/j.molcatb.2009.01.003","title":"Ureases I. Functional, catalytic and kinetic properties: A review","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"16584179","doi":"10.1021/bi052020p","title":"Catalyzed Decomposition of Urea. Molecular Dynamics Simulations of the Binding of Urea to Urease†","evidence_types":["computational experiment"]},{"pubmed_id":"17676790","doi":"10.1021/jp072323o","title":"Competitive Hydrolytic and Elimination Mechanisms in the Urease Catalyzed Decomposition of Urea","evidence_types":["computational experiment"]}]},{"mechanism_id":3,"is_detailed":true,"mechanism_text":"Proposed by Benini et al. for Bacillus pasteurii enzyme, urea binds in a bidentate manner with its carbonyl oxygen bound to Ni1 and one of the amino group bound to Ni2, thus replacing three water moieties, leaving only the bridging hydroxide. This hydroxide attacks urea to give the tetrahedral transition state leading to formation of ammonia and carbamate.","rating":2,"components_summary":"reaction occurs outside the enzyme, proton transfer, inferred reaction step, unimolecular elimination by the conjugate base, intramolecular elimination","steps":[{"step_id":1,"description":"Urea binds in a bindentate manner, displacing the active site water molecules. The bridging hydroxide group initiates a nucleophillic attack on the carbamyl carbon, forming a tetrahedral intermediate.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.3.1","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.3.1.mrv"},{"step_id":2,"description":"The unbound NH2 group of the tetrahedral intermediate deprotonates His320.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.3.2","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.3.2.mrv"},{"step_id":3,"description":"The tetrahedral intermediate collapses, eliminating ammonia. The products then dissociate from the active site, replaced by water molecules.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.3.3","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.3.3.mrv"},{"step_id":4,"description":"In an inferred step, His320 abstracts a proton from one of the incoming water molecules to regenerate the enzyme's active site.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.3.4","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.3.4.mrv"},{"step_id":5,"description":"The carbamate product rearranges outside of the active site to produce carbon dioxide and ammonia.","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.3.5","is_product":false,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.3.5.mrv"},{"step_id":6,"description":"Products","figure":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes_svg/Step_macie.87.3.6","is_product":true,"marvin_xml":"www.ebi.ac.uk/thornton-srv/m-csa/media/schemes/Step_macie.87.3.6.mrv"}],"references":[{"pubmed_id":"10368287","doi":"10.1016/s0969-2126(99)80026-4","title":"A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels","evidence_types":["crystallography evidence"]},{"pubmed_id":"22670767","doi":"10.1021/ja3043239","title":"Wide-Open Flaps Are Key to Urease Activity","evidence_types":["crystallography evidence"]},{"pubmed_id":"20886006","doi":"10.1155/2010/364891","title":"Computational Modeling of the Mechanism of Urease","evidence_types":["computational experiment"]},{"pubmed_id":null,"doi":"10.1016/j.molcatb.2009.01.003","title":"Ureases I. Functional, catalytic and kinetic properties: A review","evidence_types":["traceable author statement (general)"]},{"pubmed_id":"8702515","doi":"10.1074/jbc.271.31.18632","title":"Characterization of the Mononickel Metallocenter in H134A Mutant Urease","evidence_types":["required","inferred from mutant phenotype"]},{"pubmed_id":"9201965","doi":"10.1021/bi970514j","title":"Structures of Cys319 Variants and Acetohydroxamate-InhibitedKlebsiella aerogenesUrease†,‡","evidence_types":["crystallography evidence"]},{"pubmed_id":"15038715","doi":"10.1021/ja049618p","title":"Molecular Details of Urease Inhibition by Boric Acid: Insights into the Catalytic Mechanism","evidence_types":["crystallography evidence"]},{"pubmed_id":"11713685","doi":"10.1007/s007750100254","title":"Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism","evidence_types":["crystallography evidence"]}]}],"is_polymeric":false}},{"mcsa_id":88,"enzyme_name":"UDP-glucose-hexose-1-phosphate uridylyltransferase","is_reference_uniprot_id":true,"reference_uniprot_id":"P09148","url":"www.ebi.ac.uk/thornton-srv/m-csa/entry/88/","description":"Nucleotidyltransferases catalyse the covalent modification of a variety of biological molecules. These reactions are crucial for the synthesis of coenzymes, cyclic nucleotides, polynucleotides, and nucleotide sugars. These reactions involve substitutions at the R -phosphorus of a nucleotidyl donor substrate and result in displacement of a phosphoryl ester or pyrophosphate. Substrates for such reactions may include nucleoside di- or triphosphates, as well as nucleotide sugars, such as UDP-Glc. Galactose-1-phosphate uridylyltransferase (hexose-1-phosphate uridylyltransferase) catalyses the exchange of the UMP moiety between the hexose 1-phosphates of Glc and Gal and their corresponding UDP-sugar. The enzyme is distinct among nucleotidyl transferases that use phosphates as acceptor groups in that it is the only one that does not utilise nucleoside di- or triphosphates as the nucleotidyl donor substrate. The reaction is part of the Leloir pathway of galactose metabolism required for the normal equilibration of UDP-hexoses among most organisms. Deficiencies in uridylyltransferase activity culminate in the metabolic disease galactosemia, which occurs as an autosomal recessive trait.","protein":{"sequences":[{"uniprot_id":"P09148"}]},"all_ecs":["2.7.7.12"],"residues":[{"mcsa_id":88,"roles_summary":"activator","function_location_abv":"main-C","main_annotation":"","ptm":"","roles":[{"group_function":"activator","function_type":"spectator","function":"activator","emo":"EMO_00038"}],"residue_chains":[{"chain_name":"A","pdb_id":"1hxq","assembly_chain_name":"A","assembly":1,"code":"His","resid":164,"auth_resid":164,"is_reference":true,"domain_name":"A02","domain_cath_id":"3.30.428.10"}],"residue_sequences":[{"uniprot_id":"P09148","code":"His","is_reference":true,"resid":164}]},{"mcsa_id":88,"roles_summary":"metal ligand","function_location_abv":"","main_annotation":"","ptm":"","roles":[{"group_function":"metal ligand","function_type":"interaction","function":"metal ligand","emo":"EMO_00116"},{"group_function":"metal 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1-phosphate(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/58336.mol"},{"count":1,"type":"reactant","chebi_id":"58885","name":"UDP-alpha-D-glucose(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/58885.mol"},{"count":1,"type":"product","chebi_id":"58601","name":"alpha-D-glucose 1-phosphate(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/58601.mol"},{"count":1,"type":"product","chebi_id":"66914","name":"UDP-alpha-D-galactose(2-)","mol_file":"www.ebi.ac.uk/thornton-srv/m-csa/media/compound_mols/66914.mol"}],"mechanisms":[{"mechanism_id":1,"is_detailed":true,"mechanism_text":"His166 in the active site of the the enzyme in Escherichia coli attacks the alpha-phosphorus of UDP--Glc, displaces Glc-1-P, and forms the high energy covalent uridylyl-enzyme (UMP-enzyme) intermediate, which then reacts with Gal-1-P to produce UDP-Gal.","rating":3,"components_summary":"overall product formed, enzyme-substrate complex cleavage, 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