The conformation of NADH bound to inosine 5'-monophosphate dehydrogenase determined by transferred nuclear Overhauser effect spectroscopy.
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The reaction proceeds with concomitant conversion of NAD+ to NADH and is the rate-limiting step in the de novo biosynthesis of guanosine nucleotides. IMPDH is a target for numerous chemotherapeutic agents. The conformations of enzyme-bound substrates, enzyme-bound products and enzyme-bound ligands in general, are of interest for the understanding of the catalytic mechanism of the enzyme and the design of new inhibitors. Although several of the chemotherapeutic inhibitors of IMPDH are NAD+ or NADH analogues, no structural data for IMPDH-bound NAD+ (or NADH) are available. In the present work, we have used transferred nuclear Overhauser effect spectroscopy (TRNOESY) to determine the conformation of NADH bound to the active site of human type II IMPDH (IMPDH-h2). The inter-proton distances determined from TRNOESY data indicate that NADH binds to the enzyme active site in an overall extended conformation. The adenosine moiety and the nicotinamide riboside moiety are both in the anti conformation about the glycosidic bond, and both ribose rings are in approximately C4'-exo conformations. The nicotinamide amide group was found to be in a cis conformation. The anti conformation of the nicotinamide riboside moiety is in accord with the preferred conformations of several potent and selective dinucleotide inhibitors and is consistent with that implied by the stereospecificity of hydride transfer in the enzymatic reaction. The implications of this conformation for the catalytic mechanism of IMPDH-h2 are discussed.
Schalk-Hihi
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Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Zhang
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Markham
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D000241:Adenosine / Q000737:chemistry; D002236:Carbohydrate Conformation; D003198:Computer Simulation; D006801:Humans; D006859:Hydrogen; D007168:IMP Dehydrogenase / Q000737:chemistry*; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D013997:Time Factors
D009243:NAD; D006859:Hydrogen; D007168:IMP Dehydrogenase; D000241:Adenosine