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'-monophosph ate dehydrogenase (IMPDH) were investigated using the covalent modific ation reagents 6-chloroinosine 5'-monophosphate (6-Cl-IMP) and iodoace tamide. IMPDH was incubated with these reagents in the presence and ab sence of IMP, NAD, and NADH, and the activity of the enzyme for IMP de hydrogenation 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 inacti vation in the presence of the substrate IMP. It was not protected agai nst inactivation by NAD alone. Saturating concentrations of IMP and NA DH reduced the inactivation rate by about the same amount as with IMP alone. IMPDH samples labeled with 6-Cl-IMP and an unlabeled control we re alkylated with iodoacetamide, digested with trypsin, and analyzed b y HPLC-mass spectrometry (HPLC-MS). All eight cysteines of human type II IMPDH were found to exist as free sulfhydryls on the active, unlabe led form of the enzyme. At an enzyme/inactivator ratio of 1:4, only on e cysteine residue, Cys-331, was found to be covalently modified by 6- Cl-IMP. From the results of the substrate protection experiments and H PLC-MS data, it is concluded that 6-Cl-IMP binds in the IMP binding si te of IMPDH and reacts covalently with Cys-331 to form a purine ribosi de 5'-monophosphate-enzyme adduct.