Tartrate dehydrogenase-oxalate complexes: formation of a stable analog of a reaction intermediate complex.
Oxalate has been shown to form a stable complex with Mn-tartrate dehydrogenase-NADH complexes which are proposed to mimic an intermediate formed during catalytic turnover. The formation of this complex can be detected under turnover conditions, where oxalate acts as a time-dependent inhibitor, and under equilibrium conditions, where oxalate binding triggers a slow protein conformation change detectable by fluorescence spectroscopy. Both the rate constant for the change in fluorescence intensity upon oxalate binding and the magnitude of the fluoresence change show a hyperbolic dependence on oxalate concentration. The time-dependent inhibition by oxalate is not consistent with a model in which oxalate binds to enzyme-NAD; rather, it is proposed that inhibition arises from binding to enzyme-NADH. The apparent dissociation constants of oxalate from enzyme-NAD+ and the enzyme-NADH complexes are 80 and 1 microM, respectively. The fluorescence changes which accompany oxalate binding are suggested to arise from a protein conformational change which serves to sequester reactants in the active site. Consistent with this hypothesis, it was observed that although some alternative pyridine nucleotide cofactors supported the multistep tartrate dehydrogenase-catalyzed net nonoxidative decarboxylation of meso-tartrate only at drastically reduced rates, none of the intermediate hydroxypyruvate was released into solution. In addition, fluorescence anisotropy measurements were conducted to investigate the mode of NADH binding.
Beecher
B S
BS
Department of Biochemistry, University of Missouri-Columbia 65211.
Koder
R L
RL
Tipton
P A
PA
D000227:Adenine Nucleotides / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D005454:Fluorescence Polarization; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D010070:Oxalates / Q000737:chemistry*; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
D000227:Adenine Nucleotides; D010070:Oxalates; D011994:Recombinant Proteins; D009243:NAD; D008345:Manganese; D000429:Alcohol Oxidoreductases; C038481:tartrate dehydrogenase