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首页> 外文期刊>Biochemistry >PHOSPHORUS-31 NUCLEAR MAGNETIC RESONANCE STUDIES ON COENZYME BINDING AND SPECIFICITY IN GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
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PHOSPHORUS-31 NUCLEAR MAGNETIC RESONANCE STUDIES ON COENZYME BINDING AND SPECIFICITY IN GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

机译：磷酸31-磷酸甘油三酯脱氢酶中辅酶结合及特异性的Phosphorus-31核磁共振研究

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Binding of NAD(P)(+) to wild type and a series of mutants of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus designed to alter the cofactor specificity [Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., & Branlant, G. (1993) Biochemistry 21, 10178-10184] has been studied by P-31 NMR. In the mutants with the L187A and P188S substitutions, the pyrophosphate signals are split, and the upfield resonance has been assigned to the P(a) phosphate. Titration of the NADP(+) 2'-phosphate pK(a) deduced from its chemical shift shows that the electrostatic environment in the binding site is largely affected by the single point mutations. pK(a)s ranging from 7.7 for the L187A-P188S mutant to <5.7 for the D32G-L187A-P188S and D32A-L187A-P188S mutants have been observed, thus indicating that the binding of NADP(+) is modulated by the ionization state of its 2'-phosphate. In the quintuple mutant L33T-T34G-D35G-L187A-P188S, designed in comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, the 2'-phosphate has a pK(a) of 6.8. As further stabilizing interactions like hydrogen bonds or positively charged side chains would lower this pK(a), it is suggested that the 2'-phosphate ionization state of bound NADP(+) in chloroplastic GAPDH is dianionic. The NADP(+) dissociation rate constants (k(off)) of the three mutants D32G, L187A-P188S, and D326-L187A-P188S are higher at pH 6.1 than at pH 8.1 and are similar at the same pH, indicating that the difference in binding affinity between these three mutants results from the molecular recognition step or a conformational change upon binding.
机译：NAD（P）（+）与野生型和一系列嗜热脂肪芽孢杆菌糖酵解性NAD依赖性甘油醛-3-磷酸脱氢酶（GAPDH）的突变体的结合，旨在改变辅因子的特异性[Clermont，S.，Corbier，C ，Mely，Y.，Gerard，D.，Wonacott，A。和Branlant，G。（1993）Biochemistry 21，10178-10184]已经通过P-31 NMR进行了研究。在具有L187A和P188S取代的突变体中，焦磷酸盐信号被拆分，并且高场共振已分配给P（a）磷酸盐。从其化学位移推导的NADP（+）2'-磷酸pK（a）的滴定表明，结合位点的静电环境在很大程度上受单点突变的影响。 pK（a）范围从L187A-P188S突变体的7.7到D32G-L187A-P188S和D32A-L187A-P188S突变体的<5.7范围不等，因此表明NADP（+）的结合受到电离作用的调节。 2'-磷酸的状态。在五倍体突变体L33T-T34G-D35G-L187A-P188S中，与叶绿体的光合作用依赖NAD（P）的GAPDH进行了比较，其2'-磷酸盐的pK（a）为6.8。由于进一步的稳定相互作用（如氢键或带正电荷的侧链）会降低此pK（a），因此建议在叶绿体GAPDH中结合的NADP（+）的2'-磷酸离子化状态为双阴离子。三个突变体D32G，L187A-P188S和D326-L187A-P188S的NADP（+）解离速率常数（k（off））在pH 6.1时比在pH 8.1时高，并且在相同pH时相似。这三个突变体之间结合亲和力的差异是由于分子识别步骤或结合后构象变化引起的。

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