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

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 approximately 50-fold compared with the native enzyme, and the slow progressive inhibition that is revealed indicates that the hydrolysis proceeds via a branched pathway mechanism. This is consistent with the structural data that show that the water site is preserved and that Met170 occupies part of the space that is required for substrate binding. The short contacts between the substrate and the enzyme may lead to structure perturbation and inactivation.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Zawadzke LE,Chen CC,Banerjee S,Li Z,Wäsch S,Kapadia G,Moult J,Herzberg O

doi

10.1021/bi962242a

subject

Has Abstract

pub_date

1996-12-24 00:00:00

pages

16475-82

issue

51

eissn

0006-2960

issn

1520-4995

pii

bi962242a

journal_volume

35

pub_type

杂志文章

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