Kumar Ish, Pratt R F
Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Biochemistry. 2005 Aug 2;44(30):9961-70. doi: 10.1021/bi0505417.
Bacterial dd-peptidases, the targets of beta-lactam antibiotics, are believed to catalyze d-alanyl-d-alanine carboxypeptidase and transpeptidase reactions in vivo. To date, however, there have been few concerted attempts to explore the kinetic and thermodynamic specificities of the active sites of these enzymes. We have shown that the peptidoglycan-mimetic peptide, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, 1, is a very specific and reactive carboxypeptidase substrate of the Streptomyces R61 dd-peptidase [Anderson, J. W., and Pratt, R. F. (2000) Biochemistry 39, 12200-12209]. In the present paper, we explore the transpeptidation reactions of this substrate, where the enzyme catalyzes transfer of the glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl moiety to amines. These reactions are believed to occur through capture of an acyl-enzyme intermediate by amines rather than water. Experiments show that effective acyl acceptors require a carboxylate group and thus are amino acids and peptides. d(but not l)-amino acids, analogues of the leaving group of 1, are good acceptors. The effectiveness of d-alanine as an acceptor increases with pH, suggesting that the bound and reactive form of an amino acid acceptor is the free amine. Certain glycyl-l(but not d)-amino acids, such as glycyl-l-alanine and glycyl-l-phenylalanine, are also good acceptors. These molecules may resemble the N-terminus of the Streptomyces stem peptides that, presumably, are the acceptors in vivo. The acyl acceptor binding site therefore demonstrates a dual specificity. That d-alanyl-l-alanine shows little activity as an acceptor suggested that, on binding of acceptors to the enzyme, the carboxylate of d-amino acids does not overlap with the peptide carbonyl group of glycyl-l-amino acids. Molecular modeling of transpeptidation tetrahedral intermediates and products demonstrated the likely structural bases for the stereospecificity of the acceptors and the nature of the dual function acceptor binding site. For both groups of acceptors, the terminal carboxylate appeared to be anchored at the active site by interaction with Arg 285 and Thr 299.
细菌双肽酶是β-内酰胺抗生素的作用靶点，被认为在体内催化D-丙氨酰-D-丙氨酸羧肽酶和转肽酶反应。然而，迄今为止，很少有人协同努力去探索这些酶活性位点的动力学和热力学特异性。我们已经表明，肽聚糖模拟肽甘氨酰-L-α-氨基-ε-庚二酰-D-丙氨酰-D-丙氨酸（1）是链霉菌R61双肽酶非常特异且具反应性的羧肽酶底物[安德森，J.W.，和普拉特，R.F.（2000年）《生物化学》39，12200 - 12209]。在本文中，我们探索了该底物的转肽反应，即酶催化甘氨酰-L-α-氨基-ε-庚二酰-D-丙氨酰部分转移至胺类。据信这些反应是通过胺类而非水捕获酰基酶中间体而发生的。实验表明，有效的酰基受体需要一个羧基，因此是氨基酸和肽类。D（而非L）-氨基酸，即1的离去基团的类似物，是良好的受体。D-丙氨酸作为受体的有效性随pH升高而增加，这表明氨基酸受体的结合且具反应性的形式是游离胺。某些甘氨酰-L（而非D）-氨基酸，如甘氨酰-L-丙氨酸和甘氨酰-L-苯丙氨酸，也是良好的受体。这些分子可能类似于链霉菌茎肽的N端，大概在体内是受体。因此，酰基受体结合位点表现出双重特异性。D-丙氨酰-L-丙氨酸作为受体几乎没有活性，这表明在受体与酶结合时，D-氨基酸的羧基与甘氨酰-L-氨基酸的肽羰基不重叠。转肽四面体中间体和产物的分子模拟展示了受体立体特异性和双功能受体结合位点性质的可能结构基础。对于两组受体，末端羧基似乎通过与精氨酸285和苏氨酸299的相互作用而锚定在活性位点。
