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) binding on the conformation and stability of hamster type II inosine 5'-monophosphate dehydrogenase (IMPDH) have been examined. The protein in various states of ligand occupancy was compared by analyzing susceptibility to in vitro proteolysis, the degree of binding of a hydrophobic fluorescent dye, secondary structure content as determined by far-UV circular dichroism spectra, and urea-induced denaturation curves. These analysis methods revealed consistent evidence that IMPDH undergoes a local reorganization when IMP or XMP bind. NAD+ produced no such effect. 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 binding of IMP stabilizes a closed conformation that has a higher affinity for NAD+. The data also suggest the enzyme remains in the closed configuration throughout the catalytic steps and then reverts to the open conformation with XMP release, thereby consummating the enzyme cycle. Mycophenolic acid inhibition appeared to impart even greater stability. We propose that localized conformational changes occur during the normal and mycophenolic acid-inhibited reaction sequences of IMPDH.
Nimmesgern
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Vertex Pharmaceuticals Inc., Cambridge, Massachusetts 02139-4242, USA.
Fox
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Fleming
M A
MA
Thomson
J A
JA
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002942:Circular Dichroism; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D005453:Fluorescence; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009173:Mycophenolic Acid / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D007168:IMP Dehydrogenase; D010196:Pancreatic Elastase; D009173:Mycophenolic Acid