期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 131, 期 28, 页码 9780-9788出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja9027988
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资金
- National Natural Science Foundation of China [20803048]
- National Institutes of Health [R03-AI068672, R03-AI071992]
Carboxypeptidase A is a zinc-containing enzyme that cleaves the C-terminal residue in a polypeptide substrate. Despite much experimental work, there is still a significant controversy concerning its catalytic mechanism. In this study, the carboxypeptidase A-catalyzed hydrolysis of the hippuryl-L-Phe molecule (k(cat) = 17.7 +/- 0.7 s(-1)) is investigated using both density functional theory and a hybrid quantum mechanical/molecular mechanical approach. The enzymatic reaction was found to proceed via a promoted-water pathway with Glu270 serving as the general base and general acid. Free-energy calculations indicate that the first nucleophilic addition step is rate-limiting, with a barrier of 17.9 kcal/mol. Besides activating the zinc-bound water nucleophile, the zinc cofactor also serves as an electrophilic catalyst that stabilizes the substrate carbonyl oxygen during the formation of the tetrahedral intermediate. In the Michaelis complex, Arg127, rather than Zn(II), is responsible for the polarization of the substrate carbonyl and it also serves as the oxyanion hole. As a result, its mutation leads to a higher free-energy barrier, in agreement with experimental observations.
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