CATALYTIC MECHANISM OF ACTIVE-SITE SERINE BETA-LACTAMASES - ROLE OF THE CONSERVED HYDROXY GROUP OF THE LYS-THR(SER)-GLY TRIAD
CATALYTIC MECHANISM OF ACTIVE-SITE SERINE BETA-LACTAMASES - ROLE OF THE CONSERVED HYDROXY GROUP OF THE LYS-THR(SER)-GLY TRIAD
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DOI：
10.1042/bj3010485
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发表时间：
1994-07-15
影响因子：
4.1
通讯作者：
FRERE, JM
中科院分区：
文献类型：
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作者：
DUBUS, A;WILKIN, JM;FRERE, JM
通过定点突变研究了Lys-Thr（Ser）-Gly [KT（S）G]三联体的保守羟基对A类和C类β-内酰胺酶的作用。令人惊讶的是，该官能团的消失对两种酶的青霉素酶活性几乎没有影响。A类S235 A（Ser（235）--> Ala）和C类T316 V（Thr（315）-->瓦尔）突变体对头孢菌素酶活性的影响更大，但C类T316 A突变体的影响较小。研究扩展到β-内酰胺，其中青霉素的C-3或头孢菌素的C-4上的羧基已被修饰。这些突变对这些化合物的影响与未修饰的常规青霉素和头孢菌素相同。结果进行了比较与一个类似的突变体（T299 V）的链霉菌R61 DD-肽酶。对于这种酶，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，KT（S）G三联体的第二个残基上的羟基的严格保守性比β-内酰胺酶，特别是C类β-内酰胺酶更容易理解。
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 (Ser(235)--> Ala) and the class C T316V (Thr(315)--> 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.