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Catalytic Site Atlas

CSA LITERATURE entry for 1mas

E.C. namepurine nucleosidase
SpeciesCrithidia fasciculata (Protozoa)
E.C. Number (IntEnz) 3.2.2.1
CSA Homologues of 1mas1ezr,1q8f,1yoe,2mas,3b9x,
CSA Entries With UniProtID Q27546
CSA Entries With EC Number 3.2.2.1
PDBe Entry 1mas
PDBSum Entry 1mas
MACiE Entry M0039

Literature Report

IntroductionProtozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase. The enzyme is homotetrameric (4 x 34 kDa subunits) and exhibits no known allosteric properties.
The enzyme catalyses the hydrolysis of all of the commonly occurring purine and pyrimidine nucleosides, the hydrolysis forms ribose and the associated base. The enzyme has a preference for inosine and uridine as substrates. The substrate specificity and kinetic constants are consistent with HIS 241 acting as a proton donor to activate the hypoxanthine leaving group.
Although the enzyme is established as a member of the nonspecific nucleoside hydrolases, it has a unique substrate specificity. Its mechanism includes a tightly bound catalytic Ca2+.
MechansimThe proposed active centre is a relatively large cavity. The cavity is roughly cylindrical, located at the region identified as the "topological switchpoint". From modelling studies, PHE 167 seems to be in proper orientation to interact with the purine substrate in a base-stacking interaction, as typically observed in proteins which bind nucleotides. The carboxylate groups of three of four aspartate residues found in the cavity (ASP 10, ASP 14, ASP15 and ASP 242 - ASP 14 does not coordinate) along with THR 126, coordinate a divalent cation (possibly calcium).
A proposed mechanism suggests ribooxocarbenium stabilisation with weak leaving group activation. His 241 acts as an acid to protonate the N7 of the leaving purine. Ca2+ ion together with Asp10 activates a water molecule which nucleophilically attacks ribose C1'. Ca2+ ion serves to decrease the pKa of the attacking water molecule while Asp10 accept a proton from the water molecule. These leads to a ribooxocarbenium transition state which will spontaneously dissociate to form ribose and purine. The transition state is stabilised by Asn168, which electrostatically interacts with charged ribose O4'.
Reaction

Catalytic Sites for 1mas

Annotated By Reference To The Literature - Site 1 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
AspA1010macie:sideChainIt acts as an acid to deprotonate a water molecule, which acts as a nucleophile to attack ribose C1'.
HisA241241macie:sideChainIt activates the leaving group by donating a proton to purine N7.
AsnA168168macie:sideChainIt stabilises the transition state by electrostatically interacts with charged ribose O4'.

Annotated By Reference To The Literature - Site 2 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
AspB1010macie:sideChainIt acts as an acid to deprotonate a water molecule, which acts as a nucleophile to attack ribose C1'.
HisB241241macie:sideChainIt activates the leaving group by donating a proton to purine N7.
AsnB168168macie:sideChainIt stabilises the transition state by electrostatically interacts with charged ribose O4'.

Literature References

Notes:
Degano M
Trypanosomal nucleoside hydrolase. A novel mechanism from the structure with a transition-state inhibitor.
Biochemistry 1998 37 6277-6285
PubMed: 9572842
Degano M
Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata.
Biochemistry 1996 35 5971-5981
PubMed: 8634238
Gopaul DN
Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue.
Biochemistry 1996 35 5963-5970
PubMed: 8634237
Mazumder D
Computer simulations of trypanosomal nucleoside hydrolase: determination of the protonation state of the bound transition-state analogue.
J Am Chem Soc 2002 124 8825-8833
PubMed: 12137535
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