Identification of the IMP binding site in the IMP dehydrogenase from Tritrichomonas foetus.
The IMP dehydrogenase from Tritrichomonas foetus has been identified as a potential target for antitritrichomonial chemotherapy. The gene encoding this enzyme was expressed in transformed Escherichia coli, and the recombinant protein was purified to homogeneity with an average yield of 3 mg of protein per liter of bacterial culture. Kinetic characterizations verified that the recombinant enzyme is in the authentic native state. 6-Cl-IMP, an irreversible inhibitor of the enzyme, was found to protect cysteine residue 319 of the enzyme against carboxymethylation by iodoacetamide. Radiolabeled IMP was covalently bound to the enzyme during the enzyme-catalyzed reaction via the formation of a specific adduct with cysteine residue 319. It is thus postulated that the conversion of IMP to XMP catalyzed by the IMP dehydrogenase from T. foetus is mediated by a nucleophilic attack of cysteine-319 in the enzyme protein to IMP at, most likely, its 2-position to facilitate a hydride transfer to NAD, resulting in the formation of a covalent intermediate between substrate and enzyme.
Huete-Pérez
J A
JA
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Wu
J C
JC
Whitby
F G
FG
Wang
C C
CC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007291:Inosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism / Q000494:pharmacology; D016835:Tritrichomonas foetus / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013873:Thionucleotides; D007291:Inosine Monophosphate; C018488:6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate; C003449:thioinosinic acid; D007168:IMP Dehydrogenase; D014357:Trypsin; D003545:Cysteine; D007460:Iodoacetamide