Species-specific inhibition of inosine 5'-monophosphate dehydrogenase by mycophenolic acid.
IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+) to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor of mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits IMPDH by binding in the nicotinamide half of the dinucleotide site and trapping the covalent intermediate E-XMP. The MPA binding site of resistant IMPDH from the parasite Tritrichomonas foetuscontains two residues that differ from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Arg and Gln, respectively, in the human type 2 enzyme. We characterized three mutants of T. foetusIMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species selectivity of MPA. The mutation of Lys310Arg causes a 10-fold decrease in the K(i) for MPA inhibition and a 8-13-fold increase in the K(m) values for IMP and NAD(+). The mutation of Glu431Gln causes a 6-fold decrease in the K(i) for MPA. The double mutant displays a 20-fold increase in sensitivity to MPA. Pre-steady-state kinetics were performed to obtain rates of hydride transfer, NADH release, and hydrolysis of E-XMP for the mutant IMPDHs. The Lys310Arg mutation results in a 3-fold increase in the accumulation level of E-XMP, while the Glu431Gln mutation has only a minimal effect on the kinetic mechanism. These experiments show that 20 of the 450-fold difference in sensitivity between the T. foetus and human IMPDHs derive from the residues in the MPA binding site. Of this, 3-fold can be attributed to a change in kinetic mechanism. In addition, we measured MPA binding to enzyme adducts with 6-Cl-IMP and EICARMP. Neither of these adducts proved to be a good model for E-XMP.
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Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Hedstrom
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D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009173:Mycophenolic Acid / Q000494:pharmacology*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D012265:Ribonucleotides / Q000378:metabolism; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D016835:Tritrichomonas foetus / Q000201:enzymology
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; C097754:5-ethynyl-1-(5-O-phosphonoribofuranosyl)imidazole-4-carboxamide; D007291:Inosine Monophosphate; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid