EVIDENCE FOR TRANSITION-STATE STABILIZATION BY SERINE-148 IN THE CATALYTIC MECHANISM OF CHLORAMPHENICOL ACETYLTRANSFERASE

被引:50
作者
LEWENDON, A
MURRAY, IA
SHAW, WV
GIBBS, MR
LESLIE, AGW
机构
[1] UNIV LEICESTER,DEPT CHEM,LEICESTER LE1 7RH,ENGLAND
[2] UNIV LONDON IMPERIAL COLL SCI & TECHNOL,BLACKETT LAB,LONDON SW7 2BZ,ENGLAND
关键词
D O I
10.1021/bi00460a016
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148 → Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-Å resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148 → Cys mutation results in a 705-fold decrease in kcat and the Ser-148 → Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148 → Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148 → Cys or Asn). © 1990, American Chemical Society. All rights reserved.
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页码:2075 / 2080
页数:6
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