Elimination of the Hydrolytic Water Molecule in a Class A -Lactamase Mutant: Crystal Structure and Kinetics
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- 作者：Laura E. Zawadzke ; Celia C. H. Chen ; Soojay Banerjee ; Zhong Li ; Susana Wä ; sch ; Geeta Kapadia ; John Moult ; and Osnat Herzberg
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：December 24, 1996
- 年：1996
- 卷：35
- 期：51
- 页码：16475 - 16482
- 全文大小：411K
- 年卷期：v.35,no.51(December 24, 1996)
- ISSN：1520-4995
文摘
Two site-directed mutant enzymes of the class A 
-lactamase fromStaphylococcus aureusPC1 were produced with the goal of blocking the site that in the nativeenzyme is occupied by the proposedhydrolytic water molecule. The crystal structures of these twomutant enzymes, N170Q and N170M,have been determined and refined at 2.2 and 2.0 Å, respectively.They reveal that the side chain ofGln170 displaces the water molecule, whereas that of Met170 does not.In both cases, the catalytic rateswith benzylpenicillin are reduced by 104 compared with thenative enzyme. With nitrocefin, the N170Qmutant enzyme exhibits an approximately 800-fold reduced rate comparedwith the native enzyme and inaddition, a fast initial burst with stoichiometry of 1 mol of degradednitrocefin/mol of enzyme. Stopped-flow kinetic experiments establish that the rate constant of the burstis 250 s-1, a value comparablewiththe rate of acylation of the native enzyme. Two structurally basedmechanisms that explain the kineticproperties of the N170Q -lactamase are proposed, both invoking adeacylation-impaired enzyme due tothe elimination of the hydrolytic water molecule. The catalyticrate of the N170M mutant enzyme withnitrocefin is reduced by approximately 50-fold compared with the nativeenzyme, and the slow progressiveinhibition that is revealed indicates that the hydrolysis proceeds viaa branched pathway mechanism.This is consistent with the structural data that show that thewater site is preserved and that Met170occupies part of the space that is required for substrate binding.The short contacts between the substrateand the enzyme may lead to structure perturbation andinactivation.
-lactamase fromStaphylococcus aureusPC1 were produced with the goal of blocking the site that in the nativeenzyme is occupied by the proposedhydrolytic water molecule. The crystal structures of these twomutant enzymes, N170Q and N170M,have been determined and refined at 2.2 and 2.0 Å, respectively.They reveal that the side chain ofGln170 displaces the water molecule, whereas that of Met170 does not.In both cases, the catalytic rateswith benzylpenicillin are reduced by 104 compared with thenative enzyme. With nitrocefin, the N170Qmutant enzyme exhibits an approximately 800-fold reduced rate comparedwith the native enzyme and inaddition, a fast initial burst with stoichiometry of 1 mol of degradednitrocefin/mol of enzyme. Stopped-flow kinetic experiments establish that the rate constant of the burstis 250 s-1, a value comparablewiththe rate of acylation of the native enzyme. Two structurally basedmechanisms that explain the kineticproperties of the N170Q -lactamase are proposed, both invoking adeacylation-impaired enzyme due tothe elimination of the hydrolytic water molecule. The catalyticrate of the N170M mutant enzyme withnitrocefin is reduced by approximately 50-fold compared with the nativeenzyme, and the slow progressiveinhibition that is revealed indicates that the hydrolysis proceeds viaa branched pathway mechanism.This is consistent with the structural data that show that thewater site is preserved and that Met170occupies part of the space that is required for substrate binding.The short contacts between the substrateand the enzyme may lead to structure perturbation andinactivation.