Abstract
Inosine S'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-
monophosphate (IMP) to xanthosine S'-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 suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive
inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and
mycophenolic acid are 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 diphosphate
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 (a) between 0 and 1. However,
a 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.