CONFORMATIONAL-CHANGES AND STABILIZATION OF INOSINE 5'-MONOPHOSPHATE DEHYDROGENASE ASSOCIATED WITH LIGAND-BINDING AND INHIBITION BY MYCOPHENOLIC-ACID

The effects of substrate, product, and inhibitor (mycophenolic acid) b inding on the conformation and stability of hamster type II inosine 5' -monophosphate dehydrogenase (IMPDH) have been examined. The protein i n various states of ligand occupancy was compared by analyzing suscept ibility to in vitro proteolysis, the degree of binding of a hydrophobi c fluorescent dye, secondary structure content as determined by far-UV circular dichroism spectra, and urea-induced denaturation curves, The se analysis methods revealed consistent evidence that IMPDH undergoes a local reorganization when IMP or XMP bind, NAD(+) produced no such e ffect, In fact, no evidence was found for NAD(+) binding independently of IMP. It is proposed that IMPDH adopts an open conformation around its nucleotide binding sites in the absence of substrates and that bin ding of IMP stabilizes a closed conformation that has a higher affinit y for NAD(+). The data also suggest the enzyme remains in the closed c onfiguration throughout the catalytic steps and then reverts to the op en conformation with XMP release, thereby consummating the enzyme cycl e. Mycophenolic acid inhibition appeared to impart even greater stabil ity. We propose that localized conformational changes occur during the normal and mycophenolic acid-inhibited reaction sequences of IMPDH.