Probing the active site of human IMP dehydrogenase using halogenated purine riboside 5'-monophosphates and covalent modification reagents.
Active-site amino acid residues of human type II inosine 5'-monophosphate dehydrogenase (IMPDH) were investigated using the covalent modification reagents 6-chloroinosine 5'-monophosphate (6-Cl-IMP) and iodoacetamide. IMPDH was incubated with these reagents in the presence and absence of IMP, NAD, and NADH, and the activity of the enzyme for IMP dehydrogenation or 2-Cl-IMP dehalogenation was followed. IMPDH activity was rapidly lost when the enzyme was incubated with the IMP analog, 6-Cl-IMP, or with iodoacetamide. The enzyme was protected against inactivation in the presence of the substrate IMP. It was not protected against inactivation by NAD alone. Saturating concentrations of IMP and NADH reduced the inactivation rate by about the same amount as with IMP alone. IMPDH samples labeled with 6-Cl-IMP and an unlabeled control were alkylated with iodoacetamide, digested with trypsin, and analyzed by HPLC-mass spectrometry (HPLC-MS). All eight cysteines of human type II IMPDH were found to exist as free sulfhydryls on the active, unlabeled form of the enzyme. At an enzyme/inactivator ratio of 1:4, only one cysteine residue, Cys-331, was found to be covalently modified by 6-Cl-IMP. From the results of the substrate protection experiments and HPLC-MS data, it is concluded that 6-Cl-IMP binds in the IMP binding site of IMPDH and reacts covalently with Cys-331 to form a purine riboside 5'-monophosphate-enzyme adduct.
Antonino
L C
LC
Institute of Biochemistry and Cell Biology, Syntex Discovery Research, Palo Alto, California 94304.
Straub
K
K
Wu
J C
JC
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007168:IMP Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D007202:Indicators and Reagents; D007291:Inosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007460:Iodoacetamide / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D009243:NAD / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D007202:Indicators and Reagents; D009243:NAD; D007291:Inosine Monophosphate; C018488:6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate; D007168:IMP Dehydrogenase; D014357:Trypsin; D007460:Iodoacetamide