There are 66 reactions in MACiE which have alternative mechanisms noted.
| Entry Number |
Steps |
Enzyme Name |
EC Code |
PDB Code |
CATH Code |
|
M0002
| Overall Reaction | beta-lactamase (Class A)
| 3.5.2.6
| 1btl
| 3.40.710.10
|
| Comment: | Work by Mulholland et al [4] suggests an alternative mechanism in which the activating base in step 1 is Glu166 and not Lys73.
|
|
M0002
| Step 01
| beta-lactamase (Class A)
| 3.5.2.6
| 1btl
| 3.40.710.10
|
| Comment: | Work by Mulholland et al [4] suggests an alternative mechanism in which the activating base is Glu166 and not Lys73.
|
|
M0002
| Step 02
| beta-lactamase (Class A)
| 3.5.2.6
| 1btl
| 3.40.710.10
|
| Comment: | An alternative mechanism has been suggested, using computational studies, in which the protonation of the beta-lactam nitrogen occurs as the first step in the reaction as an initiation step and not as a consequence of the C-N bond cleavage [5] as shown here.
|
|
M0003
| Overall Reaction | NAD(P)H dehydrogenase (quinone)
| 1.6.5.2
| 1d4a
| 3.40.50.360
|
| Comment: | The obligatory two-electron reduction is shown here to proceed via two direct hydride transfers, although the possibility of two one electron transfers has not been definitely ruled out. However, no semiquinone intermediate has ever been observed, ruling out this alternative to some extent. This enzyme features two independent, equivalent active sites, which are located at opposite ends of the dimer interface. Residues from both monomers line the active sites, which are large cavities extending from the protein surface to the isoalloxazine rings of the FAD cofactors [2].
|
|
M0004
| Step 02
| nitrite reductase (NO-forming)
| 1.7.2.1
| 1nia
| 2.60.40.420
2.60.40.420
|
| Comment: | Water displaces the product C00533 after this step. An alternative role for His255 in this step may be to provide a positive charge in a location that assists in stabilising the transient complex of NO and water bound to the copper [1].
|
|
M0007
| Step 01
| isocitrate dehydrogenase (NADP+)
| 1.1.1.42
| 5icd
| 3.40.718.10
Unassigned
|
| Comment: | The mechanism for this step is shown as a concerted proton and hydride transfer, in which only a partial negative charge develops on the incipient keto oxygen. However, an alternative mechanism is that the proton transfer occurs first, and that the intermediate formed is the fully ionised alcholoate, which would be well stabilised by the magnesium ion [3]. Asp283B is assumed to be the base in this step as it is the closest potential base to the alpha-hydroxyl oxygen and it also appears to be in a particularly favourable geometry for proton transfer [3]. Kinetic studies have shown that Tyr160 has a role in the hydride transfer step [1].
|
|
M0013
| Step 03
| amine dehydrogenase
| 1.4.99.3
| 2bbk
| 2.60.30.10
2.130.10.10
|
| Comment: | Second step of Schiff base formation. The alternative mechanism for this step may be a base activated dehydration.
|
|
M0013
| Step 06
| amine dehydrogenase
| 1.4.99.3
| 2bbk
| 2.60.30.10
2.130.10.10
|
| Comment: | The alternative mechanism for this step may be a base activated addition of water.
|
|
M0013
| Step 10
| amine dehydrogenase
| 1.4.99.3
| 2bbk
| 2.60.30.10
2.130.10.10
|
| Comment: | The alternative mechanism for this step may be a base activated addition of water.
|
|
M0013
| Step 11
| amine dehydrogenase
| 1.4.99.3
| 2bbk
| 2.60.30.10
2.130.10.10
|
| Comment: | The alternative mechanism for this step may be a base activated deamination.
|
|
M0031
| Step 06
| thymidylate synthase
| 2.1.1.45
| 1lcb
| 3.30.572.10
|
| Comment: | There are two alternative mechanisms proposed for this hydride transfer step. The first is a single step transfer in which the Trp82 stabilises a positively charged trsnaition state. The second is that the hydride transfer may occur by two single-electron transfers with Trp82 stabilising the radical cation intermediate through long-range electrostatic effects. The precise positioning of H-C6 over the site of htydride transfer suggests that the singe step (shown here) is likely [5].
|
|
M0050
| Overall Reaction | orotidine-5'-phosphate decarboxylase
| 4.1.1.23
| 1dbt
| 3.20.20.70
|
| Comment: | There are three alternative mechanisms that have been suggested for this reaction, the zwitterion mechanism - shown here due to evidence of the crystal structure [1], the carbene mechanism and 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. However, it is not yet possible to definitively rule out any of the suggested mechanisms [1,2].
|
|
M0056
| Overall Reaction | tRNA-pseudouridine synthase I
| 5.4.99.12
| 1ze1
| Unassigned
|
| Comment: | There are two commonly proposed mechanisms for this enzyme. Based on references 2 and 3 we show the acyl-enzyme mechanism rather than the alternative Michael addition mechanism.
|
|
M0060
| Step 04
| glucosamine-6-phosphate deaminase
| 3.5.99.6
| 1dea
| 3.40.50.1360
3.40.50.1360
|
| Comment: | There are two alternative mechanism for this step. The proton from the ammonium group could be transferred to the Asp72 carboxylate. The resulting cis-enolamine then removes the proton from Asp72 to the position C1 pro-R of the intermediate which rearranges to form fructosimine 6-phosphate. The imino bond reacts with a water molecule, giving an unstable carbinol ammonium intermediate. Alternatively, shown here, the pi-bond (C1-C2) of the cis-enol-ammonium is attacked by a water molecule coming from the re-face of the intermediate (i.e. from the same side as Asp72) [1].
|
|
M0071
| Overall Reaction | uridine nucleosidase
| 3.2.2.3
| 1eug
| 3.40.470.10
|
| Comment: | Classically an acid/base mechanism has been employed but, new evidence suggests an alternative mechanism where the glycosylic bond is cleaved in a dissociative nucleophilic substitution reaction, brought about by the control of substrate conformation energy and stereoelectronic effects within the active site.
|
|
M0087
| Overall Reaction | urease
| 3.5.1.5
| 1fwj
| 3.20.20.140
2.10.150.10
3.30.280.10
2.30.40.10
|
| Comment: | The mechanism of this enzyme has been subject to debate since the early 1920s and the precise steps in catalysis remain unclear [5]. From biochemical studies, crystal structures of native, site-directed variants, and inhibitor complexes of bacterial ureases of K. aerogenes and B. pasteurii, two alternative mechanisms have been proposed. The first (relating to this entry in MACiE) is based upon the crystal structure of K. aerogenes enzyme, urea binds with its carbonyl oxygen bound to Ni1 and retaining a water molecule in the Ni2 site. Consequently, the active-site flap closes and the Ni2-bound hydroxide acts as a nucleophile and attacks the carbonyl carbon atom of the urea molecule, which is polarized by coordination to Ni1. The reaction proceeds through a tetrahedral intermediate that releases ammonia with His320 acting as a general acid. The alternative mechanism has been proposed by Benini et al [6,7,8] for B. 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. A third proposal is the elimination reaction from Barios and Lippard [11] has also been proposed on the observation of cyanic intermediates. Theoretical work by Estiu and Merz [9,10] suggests that the elimination pathway may occur in competition with the more traditionally proposed mechanisms.
|
|
M0095
| Overall Reaction | arabinose isomerase
| 5.3.1.3
| 1fui
| 3.40.275.10
3.20.14.10
3.40.50.1070
|
| Comment: | Two alternative mechanisms have been proposed for this reaction, the ene-diol mechanism in which two amino acid bases transfer the two protons via an ene-diol intermediate and a hydride-shift mechanisms, in which the hydrogen atom at C2 migrates as a hydride to C1. The crystal structure solved for this enzyme suggest the ene-diol mechanism due to the presence of the Asp361 and Glu337 which can act as the general acid/base residues [1].
|
|
M0099
| Step 01
| quinoprotein alcohol dehydrogenase
| 1.1.99.8
| 1g72
| 2.140.10.10
4.10.160.10
|
| Comment: | The alternative hydride transfer mechanism employs Glu171 as the catalytic base, abstracting the alcohol hydroxyl proton and initiating hydride transfer to the C5 carbonyl of PQQ. The aldehyde product then leaves the active site via an unknown pathway.
|
|
M0099
| Step 03
| quinoprotein alcohol dehydrogenase
| 1.1.99.8
| 1g72
| 2.140.10.10
4.10.160.10
|
| Comment: | Computer modelling studies, which support the alternative hydride mechanism suggest that Glu171 acts as a general base through a water molecule to initiate enolisation of PQQH to PQQH2.
|
|
M0102
| Overall Reaction | L-lactate dehydrogenase (cytochrome)
| 1.1.2.3
| 1fcb
| 3.20.20.70
3.10.120.10
|
| Comment: | An alternative mechanism which proceeds via a carbanion has also been proposed. However Mowat et al. [1] firmly support the mechanism shown here, i.e. the hydride transfer mechanism.
|
|
M0104
| Overall Reaction | quinoprotein glucose dehydrogenase
| 1.1.5.2
| 1c9u
| 2.120.10.30
|
| Comment: | There are three proposed alternative mechanisms, the addition-elimination reaction in which the glucose becomes covalently attached to the PQQ, the hydride transfer to the C5 of the PQQ and the hydride transfer to the C4 of the PQQ [1,3]. Crystallographic evidence [1] strongly supports the direct hydride transfer from the glucose to the C5 of the PQQ cofactor.
|
|
M0104
| Step 05
| quinoprotein glucose dehydrogenase
| 1.1.5.2
| 1c9u
| 2.120.10.30
|
| Comment: | The PQQ cofactor can be re-oxidised in a single two-electron transfer step. Alternatively, re-oxidation can be achieved in two separate one-electron transfer steps via the free radical semiquinone form. Mechanism unclear.
|
|
M0110
| Overall Reaction | D-amino-acid oxidase
| 1.4.3.3
| 1c0p
| 3.40.50.720
3.30.9.10
|
| Comment: | There are two main competing alternative mechanisms for this reaction. The carbanion mechanism in which the alpha hydrogen of the amino acid is abstracted as a proton, requiring an active site base, which was suggested upon the observation that pig kidney DAAO catalyses the elimination of halide from beta-halogenated amino acids. The other mechanism is the hydride mechanism, in which the alpha hydrogen is transferred (as a hydride) to the N5 of the flavin. The second mechanism is shown here and is supported by high resolution crystal structures, kinetic isotope effects and free energy correlation calculations [1].
|
|
M0113
| Overall Reaction | sarcosine oxidase
| 1.5.3.1
| 2gb0
| 3.50.50.60
3.30.9.10
3.30.9.10
3.50.50.60
|
| Comment: | There are three proposed mechanisms for this enzyme. The hydride transfer mechanism is already shown in entry the homologous enzyme D-amino-acid oxidase (M0110). The polar mechanism is shown here and the single electron transfer mechanism is not shown. There is no strong evidence for any of these alternative mechanisms. The mechanism by which Cys315 becomes attached to FAD involves His45 and Arg49 [1]. This covalent attachment is essential to the enzyme's activity and is thought to function by modulating the redox potential of the FAD and holding the weakly bound FAD in the active site [2].
|
|
M0122
| Overall Reaction | protein-methionine-S-oxide reductase
| 1.8.4.11
| 1fva
| 3.30.1060.10
|
| Comment: | There are two alternative proposed mechanisms for the collapse of the covalently attached intermediate. One assumes that the collapse is facilitated by proton transfer from solvent or a residue to the oxygen atom of the intermediate and the attack of Cys218 on Cys72 [2 3]. The proposal shown in this entry corresponds to that in references 1 and 4 where reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate which has been characterised by chemical probes and mass spectrometry analyses.
|
|
M0124
| Overall Reaction | cytochrome-c oxidase
| 1.9.3.1
| 1v54
| 1.20.210.10
2.60.40.420
4.10.51.10
1.20.210.10
1.10.442.10
2.60.11.10
1.25.40.40
1.20.120.80
4.10.81.10
4.10.91.10
4.10.93.10
1.10.10.140
1.10.287.90
4.10.95.10
4.10.49.10
1.10.287.70
|
| Comment: | Cytochrome c oxidase is a redox driven proton pump that utilises free energy of oxygen reduction for creation of proton gradient across the mitochondrial membrane. All protons pumped across the membrane and three out of the four substrate protons consumed per cycle are transferred through the so-called D-pathway. It leads from the conserved Asp91 under involvement of solvent molecules straight up to Ser156 and Ser157 and from there through a large presumably water-filled cavity to the conserved Glu242 [1 2 3]. The further pathway for protons consist of two chain of water molecules. One connecting Glu242 to propionate D of haem A3 via the NH group of Trp126 and one connecting Glu242 to the catalytic site of the enzyme [1 2]. One of the chemical protons presumably comes via the K-pathway. This involves Lys319 that could receive protons either from Ser255 directly or from the conserved subunit II Glu residue indirectly. It would transfer the proton via Thr316 the hydroxy group of the side chain of hame A3 to Tyr244 which is covalently cross-linked to His240 [1,3]. The effect of the cross-link would be to lower the pKa of Tyr244 making this residue a possible proton donor for O2 intermediates bound at the active site [4]. Electrons are supplied by cytochrome c via CuA - haem A - haem A3/CuB at the binuclear centre and the residues Arg438 Arg439 and Phe377 may be involved in these electron transfers [5]. The enzyme follows an alternative mechanism if an electron is available on haem A at the beginning of the catalytic cycle. In this alternative pathway steps 2-4 are reduced to a single electron transfer step followed by a proton transfer. Therefore no pumping of a proton is observed [1].
|
|
M0126
| Overall Reaction | cytochrome-c3 hydrogenase
| 1.12.2.1
| 2frv
| 1.10.645.10
1.10.645.10
3.40.50.700
4.10.480.10
|
| Comment: | Several alternative mechanisms exist for hydrogen activation by transition metal complexes. Generally these reactions proceed through either oxidative addition or sigma-bond metathesis (which is usually described as homolytic and without strong charge polarization unless R is a polar group). However hydrogen isotope exchange and computational experiments for this enzyme indicate that the H-H cleavage reaction is heterolytic [3]. Cytochrome c3, the physiological electron acceptor for this particular hydrogenase, is a tetraheme cytochrome. The hemes all have different potentials, and probably only one or two are likely to accept electrons from the enzyme under physiological conditions.
|
|
M0126
| Step 03
| cytochrome-c3 hydrogenase
| 1.12.2.1
| 2frv
| 1.10.645.10
1.10.645.10
3.40.50.700
4.10.480.10
|
| Comment: | It is possible that Glu18 acts as a proton gate, and so this de-protonation step occurs through Glu18 and thence to bulk solvent through an extended proton relay chain [1,2], alternatively, the proton may leave the active site via a water channel [4].
|
|
M0126
| Step 05
| cytochrome-c3 hydrogenase
| 1.12.2.1
| 2frv
| 1.10.645.10
1.10.645.10
3.40.50.700
4.10.480.10
|
| Comment: | It is possible that Glu18 acts as a proton gate, and so this de-protonation step occurs through Glu18 and thence to bulk solvent through an extended proton relay chain [1,2], alternatively, the proton may leave the active site via a water channel [4].
|
|
M0132
| Overall Reaction | alkanal monooxygenase (FMN-linked)
| 1.14.14.3
| 1luc
| 3.20.20.30
3.20.20.30
|
| Comment: | There are at least three alternative proposals for the decay of the FMN 4a-peroxyhemiacetal intermediate to emit light and yield the final products carboxylic acid FMN and water (Steps 4-7). A Baeyer-Villiger mechanism involving a hydride transfer from the intermediate. A mechanistic proposal involving a rate-limiting electron transfer to an intermediate dioxirane. And finally a modified version of the chemically initiated electron exchange luminescence which predicts that the oxidation potential of the flavin should affect the rate of bioluminescence reaction and found to be so in experiments with substituted FMN analogs [4]. Mutational studies have shown that residues Phe46A Phe49A Phe114A Phe117A and Phe261A are critical to the luciferase activity due to the fact that their bulky and hydrophobic nature allow shielding of the critical intermediates from exposure to medium [3 6]. The torsional flexibility of Gly275A in a conserved loop has also been shown to be critical to luciferase activity [3].
|
|
M0133
| Step 04
| camphor 5-monooxygenase
| 1.14.15.1
| 1yrc
| 1.10.630.10
|
| Comment: | There are three alternative proton sources which are Glu366, Asp251 (shown here) and the propionate side chains of the heme (there is no direct water channel connecting these with the FeOOH group since camphor blocks the channel). Computational studies by Zheng et al. [3] have shown that the Asp251 channel is favoured over the Glu366 channel and proceeds via an alternate mechanism (to the traditional proton transfer mechanism involving the Glu366) in which the "push" effect of the thiolate ligand and the "pull" effect of the Arg186 combine to facilitate a concomitant electron and proton transfer that yields the products of this reaction. They have also shown that the pKa of Asp251 is estimated at 6.3, and thus Asp251 is approximately 20% protonated at pH 7 [3]. The overall conversion in this step can be viewed as one concerted process, i.e., and initial homolytic O-O bond cleavage triggering concomitant proton and electron transfer [3].
|
|
M0135
| Overall Reaction | peptidylglycine monooxygenase
| 1.14.17.3
| 1sdw
| 2.60.120.310
2.60.120.230
|
| Comment: | There are several different alternative proposals for this mechanism. The two literature references cited propose very similar mechanisms. The mechanism depicted here corresponds to the one given by Chen et al. [1].
|
|
M0139
| Step 01
| xanthine dehydrogenase
| 1.17.1.4
| 1v97
| Unassigned
|
| Comment: | An alternative mechanism has been proposed for the formation of the first intermediate in which the mechanism of the reaction proceeds via individual one-electron steps (rather than the obligatory two-electron chemistry of a nucleophilic attack mechanism). However the lack of inverse relationship between the one electron reduction potential for the purine substrates and the rate of catalysis suggests that the mechanism proceeds via the nucleophilic attack [2,3]. The Mo-O-C bond in the product of this step has been crystallographically proven which rules out the alternative of addition of the C8-H bond across the Mo=S group of the molybdenum centre, followed by oxygen insertion to yield an intermediate with a direct Mo-C bond [1]. Computational studies [5] suggest that this step occurs in a concerted manner, rather than an alternative step-wise manner, and that the catalytic base also serves to help stabilise the transition state.
|
|
M0141
| Overall Reaction | 4-cresol dehydrogenase (hydroxylating)
| 1.17.99.1
| 1dii
| 3.40.462.10
3.30.465.10
1.10.45.10
1.10.760.10
1.10.45.10
1.10.760.10
3.30.465.10
|
| Comment: | Work by Cunane et al. [1] suggests that there are a number of alternative proton relay pathways as well as two alternative catalytic bases (Tyr95 or Tyr473). The species which may be involved in the proton pathways are: two buried water molecules Glu427 Glu380 Tyr172 Tyr367 Arg368 Ser289 and Glu286 (which is exposed to the surface). For simplicity here we only considered the shortest pathway i.e. that including Glu380 Tyr367 a water molecule and Glu286. Arg474 stabilises the negative charge that develops at the N1/O2 locus in the hydroquinone and semiquinone forms of flavin during catalysis. Arg474 is linked to Asp167 which in turn is linked to Arg512 to form a hydrogen bonding network that may increase the electropositivity of Arg474 and enhance its ability to stabilise the anionic hydroquinone and semiquinone forms of the flavin [1].
|
|
M0144
| Step 04
| arsenite oxidase
| 1.20.98.1
| 1g8k
| 3.40.228.10
2.102.10.10
Unassigned
2.40.40.20
3.40.228.10
2.102.10.10
3.40.50.740
Unassigned
|
| Comment: | In cyclic voltammetry experiments both oxidation and reduction peaks are narrow and it was concluded that they arise from the cooperative transfer of two electrons for both the oxidation and the reduction of the molybdenum centres of arsenite reductase. The presence of two Fe-S clusters as auxiliary electron transport sites makes it easier for arsenite oxidase to undergo cooperative two-electron transfer by providing ready storage for electrons in the absence of a Mo(V) intermediate [3]. There are several alternative electron-transfer pathways from the pyrazine system to the [3Fe-4S] cluster (FS35005 in the crystal structure). The shortest is from the pterin N8 atom to the carbonyl O atom of Ser238 and then through space to SG of Cys24. A longer pathway mediated only by covalent and hydrogen bonds, is from the NH2 group via the carbonyl O of Asn704 to the side chain of Arg101 and then to the S1 atom of the cluster. Electron transfer from the [3Fe-4S] cluster to the [2Fe-2S] cluster (FES5006 in the crystal structure) is likewise partly mediated by hydrogen bonds. In this case, the shortest path is from the S3 atom of the [3Fe-4S] cluster to the amide N atom of Ser99, through the conjugated amide bond to the carbonyl O atom of Ser98, from the O atom across the subunit interface to the imidazole NE2 atom of the Rieske cluster binding residue His62B and finally to the [2Fe-2S] FE1 atom. Other potential pathways are from S1 or S3 of the [3Fe-4S] cluster to OG of Ser99, through bonds to the amide group, and then to the [2Fe-2S] site as before [1].
|
|
M0146
| Overall Reaction | pyrogallol hydroxytransferase
| 1.97.1.2
| 1ti6
| Unassigned
3.30.70.20
2.60.40.10
|
| Comment: | There are three alternative proposals for the catalytic mechanism. Two mechanisms function without co-substrate involve a rotation of the substrate in the active site and the transferred hydroxyl stems from the solvent. The mechanism shown here includes 1,2,3,5-tetrahydroxybenzene as co-substrate through a diphenylether intermediate and therefore the transferred hydroxyl does not originate from solvent. The 3D structure of the enzyme supports the participation of a co-substrate [1]. The role served by Tyr404A could also be served by Cys557A.
|
|
M0156
| Overall Reaction | coenzyme-B sulfoethylthiotransferase
| 2.8.4.1
| 1mro
| 3.30.70.470
1.20.840.10
3.30.70.470
3.90.320.20
3.90.390.10
|
| Comment: | There are at least two proposed alternative mechanisms for this reaction. The mechanism depicted here is better supported by stereochemical and energetic factors [1,3]. However it should be noted that it is still unclear which mechanism is the correct one [4]. The second mechanism proposed assumes that the first step is the attack by the Ni(I) on the thioether sulfur of methyl-coenzyme M. This yields a free methyl radical which reacts with the thiol group of coenzyme B to give methane and the coenzyme B thiyl radical [2]. Ni is from the hydroporphinoid nickel complex coenzyme Factor 430.
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|
M0163
| Overall Reaction | calf thymus ribonuclease H
| 3.1.26.4
| 1rdd
| 3.30.420.10
|
| Comment: | Two alternative mechanisms have been proposed for this enzyme. One is a two-metal-ion mechanism where one of the two metal ions activates the attacking hydroxide ion and the other is a general acid-base mechanism where an amino acid residue fulfils this role. NMR and kinetic studies suggest only one metal binds to this protein and that the protein is inactivated by subsequent metal binding events. This makes the general acid base mechanism more plausible [1]. Asp134 holds the nucleophilic water molecule, Glu48 anchors the water molecule that acts as a general acid.
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|
M0166
| Overall Reaction | leukotriene-A4 hydrolase
| 3.3.2.6
| 1hs6
| Unassigned
|
| Comment: | Leukotriene-A4 hydrolase is a bifunctional zinc metalloenzyme which performs epoxide hydrolysis (shown here) and also shows aminopeptidase activity from a common active site. The mechanism shown here is one of two possibilities. The alternative mechanism suggested includes the formation of an ester intermediate. In this scheme the zinc alone activates and opens the epoxide and a carboxylate of Glu271 attacks the substrate at C6 to form an ester intermediate. In a concerted SN2' reaction this ester can then be attacked by a hydroxo group at C12 and a negative charge can move along the conjugated triene system eventually leading to an alkyl-oxygen cleavage. However such cleavages of esters are rare reactions although not unknown [1].
|
|
M0171
| Overall Reaction | carboxypeptidase A
| 3.4.17.1
| 1m4l
| 3.40.630.10
|
| Comment: | Several alternative catalytic pathways have been suggested for this enzyme which may be divided into two major groups. The first of which starts from direct nucleophilic attack on the peptide carbonyl by Glu270. The second pathway involves the general acid/general base Mechanism in which the water that attacks the peptide carbonyl is initially activated by the Zn/Glu270 system or by the C terminal carboxylic group of the substrate. We show the general acid/general base mechanism in which the water is activated by Zn/Glu270. This is supported although not conclusively in ref [1].
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|
M0181
| Step 01
| phosphonoacetaldehyde hydrolase
| 3.11.1.1
| 1rql
| 3.40.50.1000
1.10.150.240
3.40.50.1000
|
| Comment: | There are two kinetically equivalent routes for the formation of the dipolar intermediate (first step) which give rise to two alternative mechanisms. One in which the native state includes protonated His56 and neutral Lys53 (shown here) and other in which Lys53 is protonated and His56 is neutral. However, the extensive hydrogen bond network that incorporates the proton on the His56 ring (comprising the ring N(1)H of the His56, the backbone carbonyl of Ala45, the N(3)H hydrogen bond to Wat-120, and the hydrogen bond between Wat-120 and the sulfur atom of Met49) may serve to increase the basicity of His56 to a value greater than that of Lys53 when the enzyme is in the closed conformation. Consequently, at neutral pH, which is both the pH optimum for catalysis and the prevailing pH in the cell, His56 is charged and Lys53 is not [1].
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|
M0182
| Step 01
| methylisocitrate lyase
| 4.1.3.30
| 1mum
| 3.20.20.60
3.20.20.60
|
| Comment: | The identity of the catalytic base in this step is not clear. Four possible alternatives for this mechanism have been suggested. There are two possible water molecule candidates, one hydrogen bonded to Asp58 and the other to Glu115. Both water molecules are anchored to the magnesium cofactor. Another alternative is that the Arg158 guanidinium group may serve as a proton shuttle, however the Arg158 is already positively charged. Finally, the Tyr43 hydroxyl group which is hydrogen bonded to His113 (which is in turn hydrogen bonded to an internal water and Glu115) may also act as the base. However, a protein with similar function in the same family lacks this tyrosine histidine pair and instead has two phenylalanine residues, We show here the Asp58 alternative due to mutational studies showing the Asp58 is essential for catalytic turnover [1].
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|
M0184
| Overall Reaction | pectate lyase
| 4.2.2.2
| 1ru4
| 2.160.20.10
|
| Comment: | There are 3 alternative pathways the mechanism may proceed by: E1 E2 and E1cb. The presence of a putative catalytic base but no clear catalytic acid suggests that the favoured reaction pathway would be E1cb. This is further substantiated by the Calcium ion interaction with the substrate carboxylate at C5.
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|
M0192
| Overall Reaction | prostaglandin-E synthase
| 5.3.99.3
| 1z9h
| Unassigned
|
| Comment: | The enzyme is active in the absence of an R-SH reagent, but the catalytic activity is increased by the presence of an R-SH reagent, suggesting that a water molecule and the SH group of an R-SH bind the same site and participate in the same catalytic role. An R-SH or water molecule bound between O-eta of Tyr107 and C9 of PGH2 is polarised by forming a H-bond with Tyr107, and consequently, the SH group of R-SH or water is deprotonated at neutral pH. In reference [1] an alternative mechanism is also outlined in which Cys110 abstracts the hydrogen atom attached to C9 of the substrate and the O9-O11 bond is cleaved by acid catalysis with a water or R-SH molecule. However no evidence supports either mechanism strongly.
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|
M0201
| Overall Reaction | propionyl-CoA carboxylase
| 6.4.1.3
| 1xny
| 3.90.226.10
|
| Comment: | The enzyme consists of two polypeptides: an alpha-subunit containing the BC and BCCP domains and a beta-subunit corresponding to the CT domain [2]. BC catalyses carboxylation of the biotin attached to the biotin carboxyl carrier protein (BCCP) in a reaction that requires ATP Mg(II) and bicarbonate (steps 1 to 4)[1]. However no structural data is reported for this subunit thus the function of the magnesium ion and the identity of the catalytic base are unknown. CT catalyses the carboxyl transfer from biotin to propanoyl-CoA (steps 5 to 7)[1 2]. This subunit corresponds to PDB code 1xny. BTN5600 is the biotin cofactor which in the physiological enzyme is attached to a Lysine residue in the BCCP domain. An alternative acid catalysed mechanism for steps 5 to 7 should be chemically feasible and has previously been observed for Carboxybiotin Carboxyimidazolidone and Carbamates. However the required acid and base could not be identified in the active site.
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M0222
| Step 09
| fructose-bisphosphate aldolase (Class I)
| 4.1.2.13
| 2qut
| 3.20.20.70
3.20.20.70
|
| Comment: | It is possible that the proton relay occurs through Glu187, as in the second stage of this reaction. Alternatively, the lysine might take the proton directly from the hydroxyl group.
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M0223
| Overall Reaction | pyruvate carboxylase
| 6.4.1.1
| 2qf7
| 3.20.20.70
3.30.470.20
3.40.50.20
2.40.50.100
3.10.600.10
|
| Comment: | The metal ion can be Manganese or Zinc. This mechanism is putative and there are still many alternative mechanisms [3]
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M0245
| Overall Reaction | tyrosine 2,3-aminomutase
| 5.4.3.6
| 2rjr
| Unassigned
|
| Comment: | There are two alternative mechanisms suggested for the action of the highly electrophilic MIO cofactor. The amino-MIO adduct path is shown here as crystal structures have suggested that this is the most likely path [1,2]. However, the Friedel-Crafts-type mechanism, in which the MIO is proposed to react with the aromatic ring of the substrate, is not totally excluded [1].
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M0246
| Overall Reaction | receptor protein-tyrosine kinase
| 2.7.10.1
| 1ir3
| 1.10.510.10
3.30.200.20
|
| Comment: | Insulin receptor tyrosine kinase is thought to have a restricted range of protein substrates, with a consensus YMXM phosphorylation motif having been defined [1]. There are two alternative mechanisms suggested for this enzyme, and there is still some debate as to whether this enzyme works via the associative (SN2-type) mechanism or the dissociative (SN1-type) mechanism. However, experimental and QM/MM studies, as well as crystallographic evidence, seems to support the dissociative mechanism [1,2,3].
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M0254
| Step 05
| squalene-hopene cyclase
| 5.4.99.17
| 1h3b
| 1.50.10.20
1.50.10.20
|
| Comment: | Finally, the enzyme reaction is terminated by either regiospecific elimination of the Z-methyl group to the major product hop-22(29)-ene (80%), alternatively, addition of water forms the minor product hopan-22-ol (20%). It has been proposed that the water molecules are polarized by residues in the hydrogen-bonding network around Glu45 at the bottom of the active-site cavity; a polarized water molecule abstracts the proton or attacks the E-ring cation, to terminate the reaction. In fact, the product ratio of hopene and hopanol has been reported to be significantly altered in E45A and E45D mutants of A. acidocaldarius SHC [1].
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M0258
| Overall Reaction | beta-lactamase (Class B)
| 3.5.2.6
| 1sml
| 3.60.15.10
|
| Comment: | The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among beta-lactamases in that it is tetrameric. S. maltophilia has emerged as a significant hospital-derived pathogen of immunocompromised hosts such as cancer, cystic fibrosis and transplant patients. L1 is localised to the periplasm and hydrolyses carbapenem drugs, conferring antibiotic resistance. L1 is of the class 3a metallo-beta-lactamases and binds two Zn(II) ions for the hydrolytic reaction. An alternative to the mechanism stated here is that the reaction proceeds via a oxyanion intermediate, which might be stabilised by interactions with Tyr191, rather than the tetrahedral transition state to the nitrogen anion intermediate, which is stabilised by the two zinc ions [2].
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M0260
| Overall Reaction | hydroxymethylbilane synthase
| 2.5.1.61
| 1gtk
| 3.40.190.10
3.30.160.40
|
| Comment: | The enzyme works by stepwise addition of pyrrolylmethyl groups until a hexapyrrole is present at the active centre. The terminal tetrapyrrole is then hydrolysed to yield the product, leaving a cysteine-bound dipyrrole on which assembly continues. In the presence of a second enzyme, EC 4.2.1.75 uroporphyrinogen-III synthase, which is often called cosynthase, the product is cyclised to form uroporphyrinogen-III. If EC 4.2.1.75 is absent, the hydroxymethylbilane cyclises spontaneously to form uroporphyrinogen I. It is currently still unclear how the cysteine-bound dipyrrole is formed. There are two current alternative mechanisms that have been suggested. Either by a stepwise addition of porphobilinogen to the cysteine [1,2], or by addition of the preuroporphyinogen and thus the first catalytic turnover of the enzyme proceeds via the addition of only a further two porphobilinogen molecules [3].
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M0262
| Overall Reaction | trichodiene synthase
| 4.2.3.6
| 1jfg
| 1.10.600.10
1.10.600.10
|
| Comment: | The active site contour is complementary in both shape and hyrdophobicity to the productive conformer of the farnesyl substrate [1]. The three magnesium ions are bound to the enzyme in the presence of the substrate, the binding of which triggers a protein conformational change. The purpose of the protein conformational change is proposed to facilitate the departure of the pyrophosphate group of the substrate. Alternative mechanism [2] proposed in which the transformation of the intermediate bisabolyl cation to the cuprenyl cation involves a proton transfer rather than the more commonly proposed hydride transfer.
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M0267
| Step 07
| dihydrodipicolinate synthase
| 4.2.1.52
| 1dhp
| 3.20.20.70
|
| Comment: | Dobson et al. [2] have suggested that the cyclisation process may occur outside of the active site and that once addition has occurred, hydrolysis breaks the enzyme-substrate bond and the acyclic intermediate leaves the active site. In this alternative mechanism, the hydrolytic water is activated by the carbonyl backbone of Ile203
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M0269
| Step 03
| muconate cycloisomerase
| 5.5.1.1
| 1muc
| 3.20.20.120
3.30.390.10
|
| Comment: | Inferred return step. Alternatively, the enzyme may catalyse the reverse reaction, in which the Lys169 deprotonates the alpha carbon of 2,5-dihydro-5-oxofuran-2-acetate.
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M0270
| Overall Reaction | ribulose-phosphate 3-epimerase
| 5.1.3.1
| 1h1z
| 3.20.20.70
3.20.20.70
|
| Comment: | This enzyme participates in both the oxidative and reductive pentose phosphate pathways and is thus an amphibolic enzyme [2]. There is some discussion as to whether this enzyme is zinc dependent or not. No zinc dependence has been reported [2]. The zinc independent (in which a water molecule is bound in place of a zinc ion to the two histidine residues that are seen as the zinc binding ligands as well as the two catalytic aspartate residues) mechanism has issues relating to how the intermediate formed is stabilised, and it has been suggested that the three strictly conserved methionines (Met40, Met71 and Met144) act as a transient 'electrostatic cushion' [1]. However, the alternative in which the zinc ion stabilises the oxyanion formed is more attractive [2] with the methionine residues aiding in this process and ensuring a hydrophobic and thus proton free environment. The actual mechanisms (with respect to the bonds formed and cleaved) are identical between the two proposals, the only difference lies in how the intermediate is stabilised [1,2,3].
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M0271
| Overall Reaction | phosphoenolpyruvate mutase
| 5.4.2.9
| 1pym
| 3.20.20.60
|
| Comment: | There are three alternative mechanisms by which a phosphoryl transfer may proceed: (i) dissociative with a trigonal metaphosphate intermediate, (ii) concerted, associative transfer, involving a trigonal bipyramidal transition state, (iii) stepwise associative transfer (the addition-elimination pathway), involving a trigonal bipyramidal intermediate. The initial proposal had Asp58 acting as the phosphoryl carrier [1]. However, subsequent studies have suggested that the dissociative mechanism (whilst unusual) is more likely for this enzyme [2,3,4]. However, it is still unclear whether the reaction proceeds via a distinct metaphosphate intermediate, or a highly separated transition state [4].
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M0272
| Overall Reaction | isocitrate lyase
| 4.1.3.1
| 1f61
| 3.20.20.60
3.20.20.60
|
| Comment: | The exact roles of the amino acid residues and their protonation states in the resting state of the enzyme are not completely clear, there are two alternative mechanisms. The first has been suggested based on the membership of this protein to the enolase superfamily in which Cys191 acts forms a covalent bond to the glyoxylate substrate. The second (shown here) suggests that there is no covalent intermediate, and that Cys191 simply abstracts the alpha-proton from the succinate substrate. There is little direct evidence to support one mechanism over the other, save that to date there are no crystal structures which have a the glyoxylate covalently bound to Cys191.
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M0274
| Overall Reaction | pyruvate oxidase
| 1.2.3.3
| 1pow
| 3.40.50.970
|
| Comment: | An alternative mechanism involving a transient radical cation has been proposed based on the same evidence present in the cited references.
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M0274
| Step 07
| pyruvate oxidase
| 1.2.3.3
| 1pow
| 3.40.50.970
|
| Comment: | Phosphate attacks the kinetically stable anion radical adduct. Kinetics experiments have shown the presence of phosphate to enhance the rate of electron transfer [4]. The negative charge resulting from the nucleophilic attack by phosphate is thought to reduce the potential of the intermediate and therefore increase the driving force of the following electron transfer to FAD.
An alternative mechanism has been proposed in [4] which involves the homolytic fragmentation of a phosphate radical adduct.
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M0276
| Step 02
| nitrate reductase
| 1.7.99.4
| 1ogy
| 3.40.228.10
Unassigned
3.40.228.10
3.40.50.740
2.40.40.20
Unassigned
|
| Comment: | Theoretical studies have shown an inner sphere mechanism to be more favourable than a potential second sphere alternative [3].
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M0277
| Step 13
| mercury(II) reductase
| 1.16.1.1
| 1zk7
| Unassigned
|
| Comment: | Alternative mechanisms have been proposed for this step [4,5]. The C(4)a position is also able to attack at Cys141, forming an enzyme-FAD complex with loss of Hg(0). Reductive elimination initiated by the thiolate of Cys136 regenerates the cofactor.
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M0284
| Overall Reaction | thiocyanate hydrolase
| 3.5.5.8
| 2dd5
| 3.90.330.10
3.90.330.10
|
| Comment: | Thiocyanate hydrolase catalyses the degradation of thiocyanate to carbonyl sulfide and ammonia. An alternative mechanism, involving enzyme-substrate covalent intermediates has been suggested, although no evidence is available to support the existence of such species [2]. Structural studies show all three chains to surround the active site, with positive side chains projecting towards the metal centre from each of them, stabilising the negatively charged substrate and product [1].
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M0307
| Step 02
| Ubiquitin transfer cascade (E1, E2, E3)
| 6.3.2.19
| 3cmm
| Unassigned
3.10.20.90
|
| Comment: | The adenylation site is approx. 35 A from the catalytic cysteine, situated in the thio-ester formation site. A change in conformation upon adenylation is thought to bring the two reaction sites closer. This structural rearrangement is also thought to orientate residues into a catalytically active arrangement, including the postulated, although unidentified general base [1].
Structural conservation and crystallographic data suggests that Thr601A may act as a general base, both towards Cys600A of E1 and also Cys88A of E2, although little evidence is available to substantiate this alternative description further [18]. Structural modelling has indicated that a general base may not be necessary, and instead the presence of an oxyanion hole drives the reaction forward [5].
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M0308
| Overall Reaction | xylose isomerase
| 5.3.1.5
| 1xld
| 3.20.20.150
3.20.20.150
|
| Comment: | Three alternative mechanisms have been proposed for xylose isomerase. However, solvent isotope kinetic studies support a 1,2 hydride transfer mediated by the presence of divalent cationic metal cofactors, rather than an ene-diol mechanism [3].
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M0311
| Overall Reaction | phosphopyruvate hydratase
| 4.2.1.11
| 7enl
| 3.20.20.120
3.30.390.10
|
| Comment: | This enzyme catalyses the interconversion of 2-PGA and PEP in a reversible manner. In the dehydration direction (show here) both Lys345 and Glu211 (the catalytic acid/base pair) are neutral in charge [2,4]. At the end of the dehydration reaction, the residues are in the correct protonation state to perform the hydration reaction, i.e. Lys345 is positively charged and Glu211 negatively charged. It is assumed that at physiological pH that both protonation states of the enzyme coexist in reasonable proportions [4]. An alternative mechanism has been proposed in which His159 acts as the general base and Lys396 acts as the general acid [3].
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