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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1im5
Original Entry
Title:
Hydrolase
Compound:
180aa long hypothetical pyrazinamidase/nicotinamidase
Mutant:
No
UniProt/Swiss-Prot:
O58727-O58727
EC Class:
3.5.1.19
Other CSA Entries:
Overview of all sites for 1im5
Homologues of 1im5
Entries for UniProt/Swiss-Prot: O58727
Entries for EC: 3.5.1.19
Other Databases:
PDB entry: 1im5
PDBsum entry: 1im5
UniProt/Swiss-Prot: O58727
IntEnz entry: 3.5.1.19
Literature Report:
Introduction:
Pyrazinamide (PZA) is a prodrug used in the treatment of tuberculosis. PZA is converted to the active pyrazinoic acid by the bacterial PZAase enzyme;
mutations in PZAase can therefore confer PZA resistance to M. tuberculosis.

The gene product of Pyrococcus horikoshii 999 (PH999) shows PZAase and nicotinamidase activity, and extensive sequence homology to M. tuberculosis PZAase.
Mechanism:
The mechanism of PH999 can be inferred by homology to other enzymes like
Arthrobacter sp. CSHase and E. coli YcaC. The mechanism of substrate amide hydrolysis is likely to be via an acylated cysteine intermediate.

1) Asp 10 deprotonates Cys 133, activating Cys as a nucleophile.
2) The Cys 133 thiolate attacks the amide group of pyrazinamide.
3) The tetrahedral intermediate is stabilised by an oxyanion hole comprising the backbone amides of Ala 129 and Cys 133.
4) The tetrahedral state collapses to yield the acylated Cys 133. The NH2 leaving group is protonated by Asp 10 to yield ammonia.
5) Zn(II) coordinates a hydroxide ion that attacks the newly formed thioester.
6) The tetrahedral intermediate is again stabilised by the oxyanion hole.
7) A water molecule binds to Zn(II); Zn(II) acidifies the water so that Asp 10
can deprotonate it. (The resulting hydroxide ion attacks the acyl-enzyme in the next catalytic cycle.)
8) The tetrahedral intermediate collapses. Asp 10 protonates the leaving Cys 133 thiolate.

Lys 94 is likely to stabilise any charge on Cys 133 and Asp 10 during the reaction.
Sites:

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Found by:
Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ASPA 10 10Sidechain
Acid/baseResidue
Acid/baseWater
Acid/baseSubstrate
Asp 10 deprotonates Cys 133 and water, both of which act as nucleophiles during the reaction. Asp 10 transfers this proton to the leaving group, i.e. the Cys 133 thiolate, and -NH2 to give ammonia.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11714269 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 10390238 Related protein: UniProt Q50575 Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
LYSA 94 94Sidechain
ElectrostaticResidue
Lys 94 stabilises the negative charges on the Cys 133 thiolate and Asp 10 carboxylate, tuning their pKas by hydrogen bonding.
Evidence from paper Evidence concerns Evidence type
PubMed ID 10390238 Related protein: UniProt Q50575 Mutagenesis of residue
PubMed ID 11714269 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ALAA 129 129Backbone amide
ElectrostaticSubstrate
ElectrostaticTransition state
The backbone amide of Ala 129 forms part of the oxyanion hole.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11714269 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 133 133Sidechain, Backbone amide
NucleophileSubstrate
ElectrostaticSubstrate
ElectrostaticTransition state
Cys 133 is the nucleophilic cysteine which becomes acylated in the intermediate. The backbone amide is part of the oxyanion hole.
Evidence from paper Evidence concerns Evidence type
PubMed ID 1381445 Related protein: UniProt P32400 Chemical modification of residue
PubMed ID 10390238 Related protein: UniProt Q50575 Mutagenesis of residue
PubMed ID 11714269 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
ZNA 400 0
ElectrostaticWater
Zn(II) binds water and acidifies it, stabilising the more nucleophilic hydroxide for attack on the acyl-enzyme intermediate.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11714269 Current protein Ligand is essential for catalysis
PubMed ID 11714269 Current protein Residue is positioned appropriately (ligand position known)
Notes:

References:
1
Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii.
X. Du and W. Wang and R. Kim and H. Yakota and H. Nguyen and S. H. Kim
Biochemistry 40, (47) 14166-72, (2001).
11714269
2
Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution.
M. J. Romão and D. Turk and F. X. Gomis-Rüth and R. Huber and G. Schumacher and H. Möllering and L. Rüssmann
J Mol Biol 226, (4) 1111-30, (1992).
1381445
3
Characterization of new mutations in pyrazinamide-resistant strains of Mycobacterium tuberculosis and identification of conserved regions important for the catalytic activity of the pyrazinamidase PncA.
N. Lemaitre and W. Sougakoff and C. Truffot-Pernot and V. Jarlier
Antimicrob Agents Chemother 43, (7) 1761-3, (1999).
10390238
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