The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.
The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase.
复制标题
DD-肽酶的作用机制：苏氨酸-299 和-301 在链霉菌 R61 DD-肽酶中的作用。
作者：
J. Wilkin;A. Dubus;B. Joris;J. Frère
链霉菌 R61 DD 肽酶中残基 Thr299 和 Thr301 的侧链已通过定点诱变进行了修饰。这些氨基酸是形成活性位点空腔壁的β链的一部分。 Thr299 对应于 Lys-Thr(Ser)-Gly 三联体的第二个残基，在活性位点 β-内酰胺酶和青霉素结合蛋白 (PBP) 中高度保守。 Thr301 的修饰仅导致酶的催化和青霉素结合特性发生微小变化。对于任何特定类别的化合物，没有观察到酰化速率的选择性降低。相比之下，299 位残基的羟基缺失会产生严重受损的酶。青霉素类的灭活率降低了30-50倍，而头孢菌素类的反应受影响更大。针对肽底物的水解效率也严重降低。更令人惊讶的是，突变体完全无法催化转肽反应。因此，这些结果很容易解释 PBP 中该位置羟基化残基的保守性。
The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.