Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase
Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase
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DOI：
10.1074/jbc.272.24.15106
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发表时间：
1997-06-13
影响因子：
4.8
通讯作者：
Branlant, G
中科院分区：
文献类型：
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作者：
BoschiMuller, S;Azza, S;Branlant, G
大肠杆菌gapb编码蛋白与甘油醛-3-磷酸脱氢酶（GAPDH）具有40%以上的氨基酸同源性。大多数氨基酸参与辅因子和底物与GAP的结合；DR在gapb编码的蛋白中保守，该酶显示出有效的非磷酸化红细胞-4-磷酸脱氢酶活性(Zhao， G., Pease， A， J., Bharani， N., and Winkler， M. E.（1995）。177,2804 -2812)但具有较低的磷酸化甘油醛-3-磷酸脱氢酶活性，而GAPDN具有较高的磷酸化甘油醛-8-磷酸脱氢酶活性和较低的磷酸化红细胞-4-磷酸脱氢酶活性。为了确定造成这些差异的结构因素，我们对大肠杆菌gapb编码蛋白和嗜脂嗜热芽孢杆菌GAPDH进行了比较动力学和结合研究。gapb编码的NAD蛋白的K-D常数比GAPDH高800倍。gapb编码蛋白氧化红细胞4-磷酸的化学机制被证明是通过与Cys-149共价中间体的两步机制进行的，其速率分别与280秒（-1）和20秒（-1）的酰化和去酰化过程相关。没有观察到同位素溶剂效应，表明限速步骤不是水解。3-磷酸甘油醛的氧化速率为0.12 s(-1)，是氢化物转移的限制性反应，与4-磷酸红细胞相比效率至少低2000倍。因此，可以得出结论，只有底物的结构决定了在酰化步骤中形成三元复合物酶- nadh -硫代半缩醛产生（或不产生）氢化物转移。GAPDH氧化4-磷酸红细胞的时间为0.1 s(-1)，并且受到酰化的限制，这一结论得到了加强。步骤，虽然甘油醛3-磷酸酰化是有效的，但不是速率决定的（大于或等于800 s(-1)），用Asn取代gapb编码蛋白上的His-176，假设残基作为碱催化剂促进氢化物转移，减少40倍的甘油醛8-磷酸氧化的k（cat）。这表明，在三元配合物中，3-磷酸甘油醛的C-1原子相对于辅助因子的吡啶的非有效定位是导致催化效率低的原因。尽管3-磷酸甘油醛的氧化磷酸化证明了Pi位点是有效的，但没有观察到gapb编码的蛋白对4-磷酸红酶的磷酸化活性。因此，无机磷酸盐与ki位点的结合可能无法有效地攻击由4-磷酸红酶形成的硫酰基中间体，而水分子是水解硫酰基中间体的有效亲核试剂。与甘油醛-3-磷酸脱氢酶活性相比，这对应于去酰化步骤的激活大于或等于4.5 Kcal.mol（-1）。总之，这些结果表明GAPDH和gapb编码蛋白的活性位点之间存在细微的结构差异，这种差异可能被底物结合的结构所揭示和/或调节。这也表明蛋白质工程方法可以用于将磷酸化醛脱氢酶转化为有效的非磷酸化酶，反之亦然。
GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAP;DR are conserved in GapB-encoded protein, This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A, J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol: 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDN shows a high efficient phosphorylating glyceraldehyde-8-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity, To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The K-D constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s(-1) and 20 s(-1), respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s(-1) and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step, This conclusion is reinforced by the fact that the rats of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s(-1) and is limited by the acylation. step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (greater than or equal to 800 s(-1)), Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the k(cat) of glyceraldehyde 8-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency, No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the ki site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate, Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by greater than or equal to 4.5 Kcal.mol(-1). Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encode protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.