Kinetic and Structural Requirements for Carbapenemase Activity in GES-Type 尾-Lactamases

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• 作者：Nichole K. Stewart ; Clyde A. Smith ; Hilary Frase ; D. J. Black ; Sergei B. Vakulenko
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：54
• 期：2
• 页码：588-597
• 全文大小：473K
• ISSN：1520-4995

文摘

Carbapenems are the last resort antibiotics for treatment of life-threatening infections. The GES 尾-lactamases are important contributors to carbapenem resistance in clinical bacterial pathogens. A single amino acid difference at position 170 of the GES-1, GES-2, and GES-5 enzymes is responsible for the expansion of their substrate profile to include carbapenem antibiotics. This highlights the increasing need to understand the mechanisms by which the GES 尾-lactamases function to aid in development of novel therapeutics. We demonstrate that the catalytic efficiency of the enzymes with carbapenems meropenem, ertapenem, and doripenem progressively increases (100-fold) from GES-1 to -5, mainly due to an increase in the rate of acylation. The data reveal that while acylation is rate limiting for GES-1 and GES-2 for all three carbapenems, acylation and deacylation are indistinguishable for GES-5. The ertapenem鈥揋ES-2 crystal structure shows that only the core structure of the antibiotic interacts with the active site of the GES-2 尾-lactamase. The identical core structures of ertapenem, doripenem, and meropenem are likely responsible for the observed similarities in the kinetics with these carbapenems. The lack of a methyl group in the core structure of imipenem may provide a structural rationale for the increase in turnover of this carbapenem by the GES 尾-lactamases. Our data also show that in GES-2 an extensive hydrogen-bonding network between the acyl-enzyme complex and the active site water attenuates activation of this water molecule, which results in poor deacylation by this enzyme.