Govardhan C P, Pratt R F
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06457.
Biochemistry. 1987 Jun 16;26(12):3385-95. doi: 10.1021/bi00386a021.
Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.
已测定了一系列无环缩肽（酰基 - D - 丙氨酰 - D - 丙氨酸肽的酯类似物）在代表性的C类（阴沟肠杆菌P99）和A类（蜡样芽孢杆菌I、TEM - 2和金黄色葡萄球菌PC1）β - 内酰胺酶催化下水解的稳态动力学参数。这些底物中最佳的，也是本研究中使用最多的，是间 - [[(苯乙酰基)-甘氨酰]氧基]苯甲酸，其裂解速率可通过分光光度法跟踪。P99酶还催化了这些底物在甲醇水溶液中的甲醇解反应。对甲醇对竞争性水解和甲醇解反应动力学以及产物分布影响的定量评估，支持了一种涉及酰基酶中间体的反应机制，其形成在底物饱和条件下是速率决定步骤。考虑这些动力学参数随缩肽结构的变化，并与双环β - 内酰胺底物的类似参数进行比较，表明该酶存在多种底物结合模式。A类酶，蜡样芽孢杆菌β - 内酰胺酶I和TEM - 2β - 内酰胺酶，催化缩肽和苄青霉素的水解，但不催化甲醇解。因此，来自这两种类型底物的酰基酶对外部亲核试剂是屏蔽的；因此这种屏蔽不是青霉素酰酶中噻唑烷基直接或间接作用的结果。金黄色葡萄球菌PC1质粒的A类β - 内酰胺酶与该类的上述两个代表明显不同，因为它确实催化缩肽的甲醇解（但不催化苄青霉素的甲醇解）。甲醇解动力学表明，在饱和时脱酰化是速率决定步骤，这一结论得到了间 - [[(苯乙酰基)甘氨酰]氧基]苯甲酸酯水解过程中离去基团离去后中间体的证明的支持。因此，已表明β - 内酰胺酶催化特定缩肽的水解，其能力与D - 丙氨酰 - D - 丙氨酸羧肽酶/转肽酶相当。然而，前一种酶的不同之处在于无法裂解类似的肽。
