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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1al6
Original Entry
Title:
Lyase
Compound:
Citrate synthase
Mutant:
No
UniProt/Swiss-Prot:
P23007-CISY_CHICK
EC Class:
2.3.3.1
Other CSA Entries:
Overview of all sites for 1al6
Homologues of 1al6
Entries for UniProt/Swiss-Prot: P23007
Entries for EC: 2.3.3.1
Other Databases:
PDB entry: 1al6
PDBsum entry: 1al6
UniProt/Swiss-Prot: P23007
IntEnz entry: 2.3.3.1
Literature Report:
Introduction:
Citrate synthase catalyses the condensation between the carbonyl of oxaloacetate and the acetyl methyl group of acetyl CoA. The product of this reaction, citryl-CoA, remains tightly bound to the enzyme and is hydrolysed to citrate and CoA in a separate chemical step that nevertheless uses the same catalytic residues. Conformational changes of the enzyme during the reaction are believed to be important in the catalytic mechanism.
Mechanism:
The condensation reaction occurs via formation of an enolate intermediate that is produced on removal of a proton from the methyl group of acetyl CoA by Asp 375. Accumulation of negative charge on the carbonyl oxygen during formation of the enolate is stabilised by donation of hydrogen bonds from His 274 and from a water molecule. The enolate now acts as a nucleophile to attack the C2 carbonyl of oxaloacetate, with His 230 acting as a hydrogen bond donor to stabilise accumulation of negative charge on the C2 carbonyl oxygen during the attack.

Cleavage of the citryl CoA intermediate involves Asp 375 and His 274, with the latter functioning to stabilise negative charge on the thioester carbonyl of citryl CoA during the hydrolysis.
Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
585 0
ElectrostaticTransition state
Acts as a hydrogen bond donor to stabilise the enolate intermediate.
Evidence from paper Evidence concerns Evidence type
PubMed ID 8011640 Current protein Residue is positioned appropriately (ligand position known)
Evidence from paper listed as having "No PubMed ID available." below. Current protein Computer modelling

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
SERA 244 244Sidechain
ElectrostaticResidue
Acts as a hydrogen bond donor to His 274, increasing the acidity and hydrogen-bond donor ability of this residue towards the enolate intermediate.
Evidence from paper Evidence concerns Evidence type
Evidence from paper listed as having "No PubMed ID available." below. Current protein Computer modelling
Evidence from paper listed as having "No PubMed ID available." below. Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 274 274Sidechain
ElectrostaticTransition state
Acts as a hydrogen bond donor to stabilise accumulation of negative charge on the carbonyl of acetyl CoA during formation of the enolate intermediate. Later stabilises accumulation of negative charge on the same carbonyl oxygen atom during hydrolysis of citryl CoA.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9657685 Current protein Conservation of residue
PubMed ID 8011640 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 9657685 Related protein: UniProt P00889 Mutagenesis of residue
Evidence from paper listed as having "No PubMed ID available." below. Current protein Computer modelling

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 320 320Sidechain
ElectrostaticTransition state
Acts as a hydrogen bond donor to polarise the C2 carbony of oxaloacetate and stabilise accumulation of negative charge on the carbonyl oxygen during attack by the enolate of acetyl CoA.
Evidence from paper Evidence concerns Evidence type
PubMed ID 9657685 Current protein Conservation of residue
PubMed ID 9657685 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 9657685 Related protein: UniProt P00889 Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 375 375Sidechain
Acid/baseSubstrate
Removes a proton from the methyl group of acetyl CoA during formation of the enolate intermediate. Is also involved in the hydrolysis of citryl CoA.
Evidence from paper Evidence concerns Evidence type
PubMed ID 8011640 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 9657685 Current protein Conservation of residue
Evidence from paper listed as having "No PubMed ID available." below. Current protein Computer modelling
PubMed ID 9657685 Related protein: UniProt P00889 Mutagenesis of residue
Notes:
1. The details of the mechanism for hydrolysis of citryl CoA are not entirely clear.

2. There has been some debate regarding whether an enolate or a neutral enol is the intermediate in the reaction. The description given here assumes an enolate, as is suggested by recent theoretical calculations.
References:
1
Computational study of the citrate synthase catalysed deprotonation of acetyl-coenzyme A and fluoroacetyl-coenzyme A: demonstration of a layered quantum mechanical approach
W. Yang and D. G. Drueckhammer
J Phys Chem B, 107, 5986-5994
No PubMed ID available
2
A very short hydrogen bond provides only moderate stabilization of an enzyme-inhibitor complex of citrate synthase.
K. C. Usher and S. J. Remington and D. P. Martin and D. G. Drueckhammer
Biochemistry 33, (25) 7753-9, (1994).
8011640
3
Effects of changes in three catalytic residues on the relative stabilities of some of the intermediates and transition states in the citrate synthase reaction.
L. C. Kurz and T. Nakra and R. Stein and W. Plungkhen and M. Riley and F. Hsu and G. R. Drysdale
Biochemistry 37, (27) 9724-37, (1998).
9657685
4
Active site mutants of pig citrate synthase: effects of mutations on the enzyme catalytic and structural properties.
C. T. Evans and L. C. Kurz and S. J. Remington and P. A. Srere
Biochemistry 35, (33) 10661-72, (1996).
8718855
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