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 guani ne nucleotide biosynthesis. Mycophenolic acid (MPA) is a potent inhibitor o f mammalian IMPDHs but a poor inhibitor of microbial IMPDHs. MPA inhibits I MPDH by binding in the nicotinamide half of the dinucleotide site and trapp ing the covalent intermediate E-XMP*. The MPA binding site of resistant IMP DH from the parasite Tritrichomonas foetus contains two residues that diffe r from human IMPDH. Lys310 and Glu431 of T. foetus IMPDH are replaced by Ar g and Gin, respectively, in the human type 2 enzyme. We characterized three mutants of T, foetus IMPDH: Lys310Arg, Glu431Gln, and Lys310Arg/Glu431Gln in order to determine if these substitutions account for the species select ivity 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. P re-steady-state kinetics were performed to obtain rates of hydride transfer , NADH release, and hydrolysis of E-XMP* for the mutant IMPDHs. The Lys310A rg 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 me chanism. These experiments show that 20 of the 450-fold difference in sensi tivity between the T. foetus and human IMPDHs derive from the residues in t he MPA binding site. Of this, 3-fold can be attributed to a change in kinet ic 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*.