Zhang Guofeng, Mazurkie Andrew S, Dunaway-Mariano Debra, Allen Karen N
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Biochemistry. 2002 Nov 12;41(45):13370-7. doi: 10.1021/bi026388n.
Phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) via a Schiff base intermediate formed with Lys53. A single turnover requires binding of Pald to the active site of the core domain, closure of the cap domain containing the Lys53 over the core domain, and dissociation of the products following catalysis. The ligand binding and dissociation steps occur from the "open conformer" (domains are separated and the active site is solvent-exposed), while catalysis occurs from the "closed conformer" (domains are bound together and the active site is sequestered from solvent). To test the hypothesis that bound substrate stabilizes the closed conformer, thus facilitating catalysis, the rates of chemical modification of Lys53 in the presence and absence of inert substrate and/or product analogues were compared. Acetylation of Lys53 with 2,4-dinitrophenylacetate (DNPA) resulted in the loss of enzyme activity. The pseudo-first-order rate constant for inactivation varied with pH. The pH profile of inactivation is consistent with a pK(a) of 9.3 for Lys53. The inhibitors tungstate and vinyl sulfonate, which are known to bind to active site residues comprising the core domain, protected Lys53 from acetylation. These results are consistent with a dynamic equilibrium between the open and closed conformations of phosphonatase and the hypothesis that ligand binding stabilizes the closed conformation required for catalytic turnover.
蜡样芽孢杆菌的膦酰乙醛水解酶（膦酸酶）通过与赖氨酸53形成的席夫碱中间体催化膦酰乙醛（Pald）的水解P-C键断裂。单次周转需要Pald与核心结构域的活性位点结合，包含赖氨酸53的帽结构域在核心结构域上方闭合，以及催化后产物解离。配体结合和解离步骤发生在“开放构象体”（结构域分离且活性位点暴露于溶剂中），而催化发生在“闭合构象体”（结构域结合在一起且活性位点与溶剂隔离）。为了验证结合的底物稳定闭合构象从而促进催化这一假设，比较了在存在和不存在惰性底物和/或产物类似物的情况下赖氨酸53的化学修饰速率。用2,4-二硝基苯乙酸酯（DNPA）对赖氨酸53进行乙酰化导致酶活性丧失。失活的伪一级速率常数随pH值变化。失活的pH曲线与赖氨酸53的pK(a)为9.3一致。已知与包含核心结构域的活性位点残基结合的抑制剂钨酸盐和乙烯基磺酸盐可保护赖氨酸53不被乙酰化。这些结果与膦酸酶开放和闭合构象之间的动态平衡以及配体结合稳定催化周转所需的闭合构象这一假设一致。
