Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase.
IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.
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Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Hedstrom
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D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D003903:Deuterium; D006801:Humans; D007168:IMP Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009173:Mycophenolic Acid / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D016835:Tritrichomonas foetus / Q000201:enzymology*
D008024:Ligands; D009243:NAD; D012694:Serine; D003903:Deuterium; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid; D003545:Cysteine