Tartrate dehydrogenase-oxalate complexes: formation of a stable analog of a reaction intermediate complex.
Tartrate dehydrogenase-oxalate complexes: formation of a stable analog of a reaction intermediate complex.
复制标题
酒石酸脱氢酶-草酸复合物：反应中间复合物的稳定类似物的形成。
DOI：
10.1006/abbi.1994.1497
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发表时间：
1994
影响因子：
3.9
通讯作者：
Tipton,PA
中科院分区：
文献类型：
--
作者：
Beecher,BS;Koder,RL;Tipton,PA
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 fluorescence 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 μM, 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 ofmeso-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.
