PMID: 2109633Feb 27, 1990Paper

Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase

Biochemistry
A LewendonA G Leslie

Abstract

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 Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative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-A 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...Continue Reading

References

Mar 20, 1979·Biochemistry·J A McCammonM Karplus
May 5, 1988·Journal of Molecular Biology·A WarshelJ K Hwang
Jun 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·A G LeslieW V Shaw
Jan 1, 1987·Methods in Enzymology·T A KunkelR A Zakour
Mar 20, 1986·Journal of Molecular Biology·A G LeslieW V Shaw
Jan 1, 1983·CRC Critical Reviews in Biochemistry·W V Shaw

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Citations

Jul 25, 2003·Trends in Biochemical Sciences·Rona R Ramsay, James H Naismith
Oct 22, 2011·Journal of Chemical Information and Modeling·Hao FanAndrej Sali
Dec 14, 2004·Annals of the New York Academy of Sciences·Gerwald JoglLiang Tong
Feb 2, 2012·Protein Science : a Publication of the Protein Society·Tapan BiswasOleg V Tsodikov
Dec 6, 2008·Folia Histochemica Et Cytobiologica·Didem CosanErinc Aral
Feb 22, 1991·Proceedings. Biological Sciences·G C Russell, J R Guest
Jan 12, 2017·Nucleic Acids Research·Takahito MukaiDieter Söll
Oct 25, 2020·International Journal of Molecular Sciences·Takahito Mukai
Jan 1, 1997·Antimicrobial Agents and Chemotherapy·I A Murray, W V Shaw

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