Dubus A, Wilkin J M, Raquet X, Normark S, Frère J M
Centre d'Ingénierie des Protéines, Université de Liège, Belgium.
Biochem J. 1994 Jul 15;301 ( Pt 2)(Pt 2):485-94. doi: 10.1042/bj3010485.
The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.
通过定点诱变研究了赖氨酸-苏氨酸（丝氨酸）-甘氨酸[KT(S)G]三联体中保守羟基的作用，涉及一种A类和一种C类β-内酰胺酶。令人惊讶的是，该官能团的消失对两种酶的青霉素酶活性影响很小。对于A类S235A（丝氨酸235→丙氨酸）和C类T316V（苏氨酸315→缬氨酸）突变体，头孢菌素酶活性受到的影响要大得多，但C类T316A突变体受到的损害较小。研究扩展到了β-内酰胺类，其中青霉素C-3位或头孢菌素C-4位的羧基已被修饰。这些突变对这些化合物的影响与对未修饰的常规青霉素和头孢菌素的影响相同。将结果与用链霉菌R61 DD-肽酶的类似突变体（T299V）获得的结果进行了比较。对于这种酶，该突变也影响了与青霉素的相互作用，并严重降低了肽酶活性。因此，对于DD-肽酶和青霉素结合蛋白来说，KT(S)G三联体第二个残基上羟基的严格保守性比β-内酰胺酶，尤其是C类β-内酰胺酶，更容易理解。
