LOCAL CONFORMATIONAL-CHANGES INDUCED BY SUCCESSIVE NICOTINAMIDE ADENINE-DINUCLEOTIDE BINDING TO DISSOCIABLE TETRAMERIC D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE - QUANTITATIVE-ANALYSIS OF A 2-STEP DISSOCIATION PROCESS
LOCAL CONFORMATIONAL-CHANGES INDUCED BY SUCCESSIVE NICOTINAMIDE ADENINE-DINUCLEOTIDE BINDING TO DISSOCIABLE TETRAMERIC D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE - QUANTITATIVE-ANALYSIS OF A 2-STEP DISSOCIATION PROCESS
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DOI：
10.1021/bi00268a009
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发表时间：
1982-01-01
期刊：
影响因子：
2.9
通讯作者：
BATKE, J
中科院分区：
文献类型：
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作者：
OVADI, J;OSMAN, IRM;BATKE, J
FITC [异硫氰酸荧光素] 共价结合高达 2 mol/mol 四聚体酶不会影响猪肌肉 D-甘油醛-3-磷酸脱氢酶 (GAPD) 的酶活性和解离特性。 NAD 与脱氢酶-FITC 复合物的结合可部分恢复由染料与 apo-GAPD 结合引起的猝灭。这种现象以及由 NAD 结合引起的特征吸收差异光谱的形成，使得追踪 NAD 诱导的染料结合区域附近的局部构象变化成为可能。 NAD 引起的光谱变化的时间过程显示出双相动力学：爆发相和慢相。由于子单元之间的协作相互作用，突发相位的幅度作为 NAD 当量的函数具有 S 形形状。从荧光各向异性测量中可以得出相同的结论。在存在过量 NAD 的情况下，可以检测到缓慢的构象变化，其幅度是 NAD 浓度的函数。这种现象可归因于更多 NAD 分子与全酶的结合。慢相遵循一级动力学，速率常数取决于酶浓度。荧光染料标记的apo-GAPD和NAD饱和的GAPD的特定荧光强度和荧光各向异性也依赖于酶浓度。 NAD 结合可能引起四聚酶空间结构的重大变化，而不显着影响亚基接触表面之间的相互作用力。数据根据两步解离模型进行定量解释。
Covalent binding of FITC [fluorescein isothiocyanate] up to 2 mol/mol of tetrameric enzyme does not affect the enzymatic activity and dissociation properties of pig muscle D-glyceraldehyde-3-phosphate dehydrogenase (GAPD). The binding of NAD to dehydrogenase-FITC complex partially reverts the quenching caused by the binding of dye to apo-GAPD. This phenomenon, as well as the formation of a characteristic absorption difference spectrum caused by the binding of NAD, makes it possible to follow the NAD-induced local conformational changes near the dye-binding region. The time course of NAD-induced spectral changes shows biphasic kinetics: a burst and a slow phase. The amplitude of burst phase as a function of NAD equivalents has sigmoidal shape due to the cooperative interaction between subunits. The same conclusion could be drawn from fluorescence anisotropy measurements. In the presence of excess NAD a slow conformational change can be detected, the amplitude of which is a function of NAD concentration. This phenomenon can be attributed to the binding of further NAD molecules to the holoenzyme. The slow phase follows first-order kinetics, and the rate constant depends on enzyme concentration. The specific fluorescence intensity and the fluorescence anisotropy of fluorescent dye labeled apo-GAPD and GAPD saturated with NAD are also dependent on enzyme concentration. NAD binding may induce major changes in the steric structure of tetrameric enzyme without influencing remarkably the interacting forces between the contact surfaces of subunits. Data are quantitatively interpreted in terms of a 2-step dissociation model.