Tai C H, Nalabolu S R, Simmons J W, Jacobson T M, Cook P F
Department of Biochemistry and Molecular Biology, University of North Texas Health Sciences Center, Fort Worth 76107, USA.
Biochemistry. 1995 Sep 26;34(38):12311-22. doi: 10.1021/bi00038a027.
The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium. A general mechanism is proposed for OASS in which OAS binds with its alpha-amine unprotonated to carry out a nucleophilic attack on C4' of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the beta-elimination of acetate. The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the alpha-carbon in the beta-elimination reaction, and a proton is likely released with the acetate product. Sulfide likely binds as HS- to undergo nucleophilic attack on the alpha-aminoacrylate intermediate, followed by protonation of the alpha-carbon by the enzyme lysine. In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the beta-elimination of acetate, but this is not the case for OASS-B. The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10(-3), while the second half-reaction is practically irreversible.
为了获取有关鼠伤寒沙门氏菌O-乙酰丝氨酸巯基酶（OASS）的A和B同工酶化学机制的信息，测定了使用天然反应物和替代反应物时动力学参数对pH的依赖性。提出了一种OASS的通用机制，其中OAS以其α-胺未质子化的形式结合，对质子化席夫碱的C4'进行亲核攻击，且乙酰羰基与质子化的酶基团（或水分子）形成氢键，这有助于乙酸盐的β-消除。与活性位点吡哆醛5'-磷酸形成席夫碱连接的酶赖氨酸在β-消除反应中使α-碳去质子化，并且一个质子可能与乙酸盐产物一起释放。硫化物可能以HS-的形式结合，对α-氨基丙烯酸酯中间体进行亲核攻击，随后酶赖氨酸使α-碳质子化。在OASS-A中，HS-与协助乙酸盐β-消除的酶基团形成氢键，但OASS-B并非如此。OASS-A第一个半反应的pH无关平衡常数为1.6×10⁻³，而第二个半反应实际上是不可逆的。
