INACTIVATION OF INOSINE 5'-MONOPHOSPHATE DEHYDROGENASE BY THE ANTIVIRAL AGENT YNYL-1-BETA-D-RIBOFURANOSYLIMIDAZOLE-4-CARBOXAMIDE 5'-MONOPHOSPHATE

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting en zyme in de novo guanine nucleotide biosynthesis, IMPDH converts inosin e 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with con comitant conversion of NAD(+) to NADH. The antiviral agent ynyl-1-beta -D-ribofuranosylimidazole-4-carboxamide (EICAR) is believed to inhibit IMPDH by forming an active metabolite, the 5'-monophosphate EICARMP. The experiments reported here demonstrate that EICARMP irreversibly in activates both human type II and Escherichia coli IMPDH, IMPDH is prot ected from EICARMP inactivation by IMP, but not by NAD(+). Further, de naturation/renaturation of the EICARMP-inactivated enzyme did not rest ore enzyme activity, which indicates that EICARMP forms a covalent add uct with IMPDH. EICARMP was successfully used to titrate the active si tes of IMPDH; these experiments demonstrate that four active sites are present in an IMPDH tetramer, Matrix-assisted laser desorption ioniza tion time-of-flight (MALDI-TOF) mass spectrometry of native E. coli IM PDH established that protein translation initiates at the third ATG of the DNA sequence. Thus, the E. coli IMPDH monomer is only 488 amino a cids long and contains five instead of six cysteines. In addition, MAL DI-TOF mass spectrometry showed that EICARMP is covalently bound to Cy s-305 (Cys-331 in human type II IMPDH numbering), suggesting that Cys- 305 functions as a nucleophile in the IMPDH reaction, The inactivation of the E. coli enzyme is a single-step reaction with k(on) = 1.94 x 1 0(4) M(-1) s(-1). In contrast, the inactivation of human type II IMPDH involves a two-step mechanism where K-i = 16 mu M, k(2) = 2.7 x 10(-2 ) s(-1) and k(on) = 1.7 x 10(3) M(-1) s(-1). These results demonstrate that significant differences exist between bacterial and human IMPDH and suggest that this enzyme may be a target for antibiotic drugs.