Abstract
Inosine 5‘-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. IMPDH converts inosine 5‘-monophosphate (IMP) to xanthosine 5‘-monophosphate (XMP) with concomitant conversion of NAD+ to NADH. The antiviral agent 5-ethynyl-1-β-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 inactivates both human type II and Escherichia coli IMPDH. IMPDH is protected from EICARMP inactivation by IMP, but not by NAD+. Further, denaturation/renaturation of the EICARMP-inactivated enzyme did not restore enzyme activity, which indicates that EICARMP forms a covalent adduct with IMPDH. EICARMP was successfully used to titrate the active sites of IMPDH; these experiments demonstrate that four active sites are present in an IMPDH tetramer. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry of native E. coli IMPDH established that protein translation initiates at the third ATG of the DNA sequence. Thus, the E. coli IMPDH monomer is only 488 amino acids long and contains five instead of six cysteines. In addition, MALDI-TOF mass spectrometry showed that EICARMP is covalently bound to Cys-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 × 104 M-1 s-1. In contrast, the inactivation of human type II IMPDH involves a two-step mechanism where K i = 16 μM, k 2 = 2.7 × 10-2 s-1 and k on = 1.7 × 103 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.