Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism

Abstract

Inosine 5''-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5''-monophosphate (IMP) to xanthosine 5''-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggest that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitior versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphage ribose. NAD inhibits the IMPDH reaction at concentrations > 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (.alpha.) between 0 and 1. However, .alpha. was greater than 1 in all cases. These results indicate that TAD, mycophenolic acid, and NAD do not inhibit simply by binding to E-XMP and suggest that the mechanism must include an isomerization step either between IMP binding and NAD binding or between NADH release and XMP release.