A TRANSFER NUCLEAR OVERHAUSER EFFECT STUDY OF COENZYME BINDING TO DISTINCT SITES IN BINARY AND TERNARY COMPLEXES IN GLUTAMATE-DEHYDROGENASE
A TRANSFER NUCLEAR OVERHAUSER EFFECT STUDY OF COENZYME BINDING TO DISTINCT SITES IN BINARY AND TERNARY COMPLEXES IN GLUTAMATE-DEHYDROGENASE
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DOI：
10.1021/bi00399a062
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发表时间：
1987-12-15
期刊：
影响因子：
2.9
通讯作者：
BELL, JE
中科院分区：
文献类型：
--
作者：
BANERJEE, A;LEVY, HR;BELL, JE
在不存在二羧酸共配体的情况下，氧化型辅酶NAD以相等的亲和力与牛肝谷氨酸脱氢酶的每个亚基的两个位点结合。在戊二酸或2-氧戊二酸存在下，对一个位点的亲和力不变，但对另一个（假定为活性位点）的亲和力显著增加，现在需要两个解离常数来描述其饱和度。转移核Overhauser效应（TRNOE）的组合连同TRNOE时间依赖性的斜率的检查表明，虽然NAD在两个结合位点处以顺式构象结合，但NADP（仅与活性位点结合）以顺式-反式混合物结合。N6至N3“、N6和N2”以及N1“至N3”NOE“与NAD的存在表明，两个辅酶结合位点的位置足够接近，以允许分子间NOE "。在2-酮戊二酸存在下，其中仅有效观察到与活性位点的结合，任一辅酶的构象为顺式。使用来自TRNOE结果的距离估计的建模研究表明，烟酰胺核糖近似于3“-内构象。在这些条件下没有分子间NOE的证据表明，虽然每个亚基的活性和常规NAD位点非常接近，但每个六聚体的六个活性位点位于大于5埃的位置。apart.
The oxidized coenzyme NAD binds to two sites per subunit of bovine liver glutamate dehydrogenase with equal affinity in the absence of dicarboxylic acid coligands. In the presence of glutarate or 2-oxyglutarate, the affinity to one site is unchanged, but the affinity to the other (presumed to be the active site) is considerably increased and now requires two dissociation constants to describe its saturation. A combination of transfer nuclear Overhauser effects (TRNOE) together with an examination of the slopes of TRNOE time dependence indicates that while NAD is bound in a syn conformation at both binding sites, NADP (which binds only to the active site) is bound in a syn-anti mixture. The existence of N6 to N3'' and N6 and N2'' and N1'' to N3'' NOE''s with NAD suggests that the two coenzyme binding sites are located near enough to allow intermolecular NOE''s. In the presence of 2-oxoglutarate where only binding to the active site is effectively observed, the conformation of either coenzyme is syn. Modeling studies using the distance estimates from the TRNOE results suggest that the nicotinamide ribose approximates a 3''-endo conformation. The absence of evidence for intermolecular NOE''s under these conditions indicates that while the active and regularly NAD sites per subunit are in close proximity, the six active sites per hexamer are located greater than 5 .ANG. apart.