Bernstein N J, Pratt R F
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Biochemistry. 1999 Aug 10;38(32):10499-510. doi: 10.1021/bi990428e.
beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.
β-内酰胺抗生素通常被认为是通过与D-丙氨酰-D-丙氨酸肽相似而抑制其靶标酶——细菌细胞壁合成所需的DD-肽酶。尽管后者的有利构象在主链构象方面在结构上与β-内酰胺类相似，但一个显著的差异是细胞壁肽的倒数第二个丙氨酸残基上存在一个D-甲基取代基。经典的β-内酰胺抗生素在相应位置是一个氢原子。在从DD-肽酶进化出β-内酰胺酶的过程中，似乎这个D-甲基会被淘汰，以确保前者的酶能够水解β-内酰胺而不是肽。在本文中，研究了倒数第二个D-丙氨酸残基（与甘氨酸残基相对）在当今DD-肽酶和β-内酰胺酶的肽底物中的作用。肽N-（苯乙酰基）-D-丙氨酰-D-苯丙氨酸和N-（苯乙酰基）甘氨酰-D-苯丙氨酸被用作针对链霉菌R61的DD-肽酶和阴沟肠杆菌P99结构上非常相似的C类β-内酰胺酶的测试对。测定了这两种底物在两种酶作用下的周转动力学。为了量化酰基-酶中间体的分配，还研究了同源的一对缩肽被D-苯丙氨酸进行氨解的情况。因此，构建了两种酶催化两种肽周转的自由能-反应坐标图。这些图谱的比较表明，在催化的所有阶段（酰基-酶以及酰化和去酰化过渡态），DD-肽酶都更倾向于D-甲基而不是氢，而β-内酰胺酶仅在肽酰化过渡态排斥D-甲基。显然发生了β-内酰胺酶使甲基均匀解离的进化过程。通过酰化四面体中间体的计算模型从结构上探究了这些结果。确定了DD-肽酶上有一个甲基口袋，而在β-内酰胺酶上则不太有利。离去基团——末端D-丙氨酸残基与这两种酶的相互作用很有趣，因为似乎不同的带正电荷的活性位点残基与羧酸盐直接相关，在β-内酰胺酶中是赖氨酸315，在DD-肽酶中是精氨酸285（而不是组氨酸298）。分析了肽的倒数第二个残基上较大取代基，特别是双环β-内酰胺中存在的杂环取代基所带来的问题。模型的定性和定量分析支持了倒数第二个D-丙氨酸在β-内酰胺酶进化中所提出的重要性。
