Elimination of the hydrolytic water molecule in a class A beta-lactamase mutant: crystal structure and kinetics

Biochemistry
L E ZawadzkeO Herzberg

Abstract

Two site-directed mutant enzymes of the class A beta-lactamase from Staphylococcus aureus PC1 were produced with the goal of blocking the site that in the native enzyme is occupied by the proposed hydrolytic water molecule. The crystal structures of these two mutant enzymes, N170Q and N170M, have been determined and refined at 2.2 and 2.0 A, respectively. They reveal that the side chain of Gln 170 displaces the water molecule, whereas that of Met170 does not. In both cases, the catalytic rates with benzylpenicillin are reduced by 10(4) compared with the native enzyme. With nitrocefin, the N170Q mutant enzyme exhibits an approximately 800-fold reduced rate compared with the native enzyme and in addition, a fast initial burst with stoichiometry of 1 mol of degraded nitrocefin/mol of enzyme. Stopped-flow kinetic experiments establish that the rate constant of the burst is 250 s-1, a value comparable with the rate of acylation of the native enzyme. Two structurally based mechanisms that explain the kinetic properties of the N170Q beta-lactamase are proposed, both invoking a deacylation-impaired enzyme due to the elimination of the hydrolytic water molecule. The catalytic rate of the N170M mutant enzyme with nitrocefin is reduced by...Continue Reading

References

Oct 1, 1991·The Biochemical Journal·J Lamotte-BrasseurJ M Ghuysen
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Feb 22, 1990·European Journal of Biochemistry·K TsukamotoT Sawai
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Citations

Jun 5, 2002·Biochimica Et Biophysica Acta·Miglena E StefanovaWilliam G Gutheil
Jul 31, 2013·Acta Crystallographica. Section D, Biological Crystallography·Karin ValegårdMichael A McDonough
Feb 27, 1999·Protein Science : a Publication of the Protein Society·J Lamotte-BrasseurR C Wade
Aug 19, 2007·Journal of Molecular Microbiology and Biotechnology·Konanani J RashamuseDon A Cowan
Nov 2, 2006·Biological & Pharmaceutical Bulletin·Masayuki HataTyuji Hoshino
May 22, 2007·Journal of Molecular Biology·Seiji NegoroYoshiki Higuchi

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