BINDING OF REDUCED NICOTINAMIDE ADENINE-DINUCLEOTIDE TO CITRATE SYNTHASE OF ESCHERICHIA-COLI-K12
BINDING OF REDUCED NICOTINAMIDE ADENINE-DINUCLEOTIDE TO CITRATE SYNTHASE OF ESCHERICHIA-COLI-K12
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DOI：
10.1021/bi00646a017
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
TONG, EK
中科院分区：
文献类型：
--
作者：
DUCKWORTH, HW;TONG, EK
大肠杆菌柠檬酸合酶能增强其变构抑制剂NADH的荧光，并使辅酶的发射峰由457 nm移至428 nm。这些效应已被用于测量在各种条件下NADH与该酶的结合。在pH 6.2时，NADH-柠檬酸合酶复合物的解离常数约为0.28 µ，但在碱性pH时会增加，似乎结合取决于pAla约为7.05的基团的质子化。在6.2-8.7的pH范围内，每个柠檬酸合酶亚基的结合位点的数目从约0.65减少到约0.25。该转变的中点在约pH 7.7处，并且它可能是已知在该pH范围内发生的酶的部分解聚的一个反映。凝胶过滤法已被用来验证荧光增强技术准确地揭示了所有的NADH分子结合的酶在感兴趣的浓度范围内。在pH7.8时，NAD+和NADP+是弱的竞争性抑制剂（K-，值大于1 mM），而5 '-AMP和3GAMP则表现出较强的抑制作用，K_i值分别为83±5和65±4 μ。底物之一乙酰辅酶A和激活剂KCl也以弱协同方式抑制结合。所有这些影响
Citrate synthase from Escherichia coli en-hances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the bind-ing of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase com-plex is about 0.28 µ at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pAla of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this tran-sition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitiveinhibitors of NADH bind-ing at pH 7.8 (K-, values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3GAMP, with K, values of 83±5 and 65±4 µ, respectively. Acetyl-CoA, one of the substrates, and KC1, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects