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Catalytic Site Atlas

CSA LITERATURE entry for 2bif

E.C. name6-phosphofructo-2-kinase
SpeciesRattus norvegicus (Rat)
E.C. Number (IntEnz) 2.7.1.105
CSA Homologues of 2bif1bif,1c7z,1c80,1c81,1fbt,1k6m,3bif,3e9e,
CSA Entries With UniProtID P25114
CSA Entries With EC Number 2.7.1.105
PDBe Entry 2bif
PDBSum Entry 2bif
MACiE Entry 2bif

Literature Report

IntroductionFructose-6-phosphate-2-kinase/fructose-2,6-biphosphatase is a bifunctional enzyme. It is responsible to regulate the concentration of fructose-2,6-phosphate (Fru-2,6-P2), a potent physiological activator of phosphofructose kinase and an inhibitor of fructose-1,6-biphosphatase. Therefore, Fru-6-P,2-kinase/Fru-2,6-Pase is important in glucose homeostasis.
The fructose-2,6-biphosphatase domain has been shown to be structurally and functionally homologous to phosphoglycerate mutase. It catalyses its reaction via a phosphoenzyme intermediate which utilises an active site histidine as a nucleophilic phosphoacceptor.
MechansimThe catalytic reaction proceeds in two steps: 1)Formation of the phosphoryl-histidine intermediate and release of fructose 6-phosphate; and 2) hydrolysis of the phosphoenzyme intermediate.
One proposed mechanism suggests that His236 acts as a nucleophile to attack the phosphate, leading to a pentacovalent transition state, which the excess negative charge is stabilised by Arg255, Asn262, Arg305 and His390. A protonated Glu325 donates a proton to the leaving Fru-6-P and then the ionised Glu325 activates a water nucleophile to attack the phosphohistidine intermediate and restore the enzyme.
Reaction

Catalytic Sites for 2bif

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GluA325326macie:sideChainIt acts as an acid to protonate the leaving Fru-6-P and acts as a base to activate a water nucleophile, which attack the phosphohistidine intermediate to restore the enzyme.
AsnA262263macie:sideChainIt interacts with the 2-phosphate of Fru-2,6-P2 and stabilises the transition state.
HisA390391macie:sideChainIt interacts with the 2-phosphate of Fru-2,6-P2 and stabilises the transition state.
ArgA255256macie:sideChainIt stabilises the transition state.
ArgA305306macie:sideChainIt neutralises the excess negative charge developed on the phosphate of Fru-2,6-P2 during the transfer to His 256, hence stabilises the transition state.
AlaA256257macie:sideChainIt acts as a nucleophile to attack the target phosphate of the substrate, forming a phosphoenzyme intermediate.

Annotated By Reference To The Literature - Site 2 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
GluB325326macie:sideChainIt acts as an acid to protonate the leaving Fru-6-P and acts as a base to activate a water nucleophile, which attack the phosphohistidine intermediate to restore the enzyme.
AsnB262263macie:sideChainIt interacts with the 2-phosphate of Fru-2,6-P2 and stabilises the transition state.
HisB390391macie:sideChainIt interacts with the 2-phosphate of Fru-2,6-P2 and stabilises the transition state.
ArgB255256macie:sideChainIt stabilises the transition state.
ArgB305306macie:sideChainIt neutralises the excess negative charge developed on the phosphate of Fru-2,6-P2 during the transfer to His 256, hence stabilises the transition state.
AlaB256257macie:sideChainIt acts as a nucleophile to attack the target phosphate of the substrate, forming a phosphoenzyme intermediate.

Literature References

Notes:
Tauler A
Hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Use of site-directed mutagenesis to evaluate the roles of His-258 and His-392 in catalysis.
J Biol Chem 1990 265 15617-15622
PubMed: 2168419
Pilkis SJ
6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase from rat liver.
J Biol Chem 1983 258 6135-6141
PubMed: 6304027
Pilkis SJ
Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.
J Biol Chem 1984 259 949-958
PubMed: 6319392
Lin K
Arg-257 and Arg-307 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase bind the C-2 phospho group of fructose-2,6-bisphosphate in the fructose-2,6-bisphosphatase domain.
J Biol Chem 1992 267 19163-19171
PubMed: 1326547
Yuen MH
Crystal structure of the H256A mutant of rat testis fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase. Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities.
J Biol Chem 1999 274 2176-2184
PubMed: 9890980
Sakurai M
Glutamate 325 is a general acid-base catalyst in the reaction catalyzed by fructose-2,6-bisphosphatase.
Biochemistry 2000 39 16238-16243
PubMed: 11123954
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