Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction
Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction
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DOI：
10.1021/bi047479i
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发表时间：
2005-04-12
期刊：
影响因子：
2.9
通讯作者：
Cook, PF
中科院分区：
文献类型：
--
作者：
Rabeh, WM;Alguindigue, SS;Cook, PF
O-乙酰丝氨酸硫化氢解酶（OASS）催化肠道细菌和植物半胱氨酸生物合成途径的最后一步，用无机二硫化物取代O-乙酰-L-丝氨酸（OAS）的β-乙酰氧基。巯基水解酶反应的前半部分，α-氨基丙烯酸酯中间体的形成，限制了总反应速率，而在后半部分反应中，以二硫化物为底物，化学被认为是扩散限制的。为了表征第二半反应，使用天然底物二硫化物和许多亲核类似物在pH 6.0-9.5范围内测量α-氨基丙烯酸酯中间体消失的伪一级速率常数的pH依赖性。对于pK（a）> 7的底物，速率是pH依赖性的，而对于pK（a）< 7的底物，速率常数是pH无关性的，这表明底物和酶基团的pK（a）s在反应的这一半中是重要的。在D2 O中，在低pD值下，氨基酸外部席夫碱被捕获，而在H2O中，反应通过释放氨基酸产物进行，这可能是所有亲核反应物的限速。产生了许多新的β-取代的氨基酸，并通过H-1 NMR光谱进行了表征。
O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-L-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D2O, at low pD values, the amino acid external Schiff base is trapped, while in H2O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by H-1 NMR spectroscopy.