The catalytic efficiency (k_cat/K_m) of the class A β-lactamase Toho-1 correlates with the thermal stability of its catalytic intermediate analog

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• 作者：Yasushi Nitanai ; Tatsuro Shimamura ; Takuro Uchiyama ; Yoshikazu Ishii ; Michiyo Takehira ; Katsuhide Yutani ; Hiroshi Matsuzawa ; Masashi Miyano
• 关键词：CTX-M-type β ; -lactamase ; Toho-1 ; Acyl-intermediate ; Thermal stability ; Differential scanning calorimetry ; β ; -lactam antibiotics
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2010
• 出版时间：April 2010
• 年：2010
• 卷：1804
• 期：4
• 页码：684-691
• 全文大小：755 K

文摘

The extended-spectrum β-lactamases are associated with antibiotic resistance. Toho-1 R274N/R276N, a Class A β-lactamase of CTX-M-type, efficiently hydrolyzes first generationcephalosporins (for example, cephalothin), in addition to cefotaxime, a third generation cephalosporin. However, this enzyme only marginally hydrolyzes the third generation cephalosporin ceftazidime, and the monobactam aztreonam. The deacylation defectiveness of the mutant Toho-1 E166A/R274N/R276N, which lacks the deacylation activity, results in the accumulation of the complex of an acylated-enzyme intermediate analog. For drug design, it would be useful if a quantitative prediction of a catalytic property were available without the need of enzymatic measurements. Therefore, we examined whether there is a correlation between the thermal stability of a catalytic intermediate (analog) and its kinetic parameters. First we measured the hydrolytic kinetics of the 14 species of β-lactam antibiotics by Toho-1 R274N/R276N, and also measured the thermal stability of the accumulated acyl-intermediates of Toho-1 E166A/R274N/R276 by differential scanning calorimetry. Here we report the correlation of these parameters. The logarithm of the catalytic efficiency for Toho-1 R274N/R276N, log(k_cat/K_m) exhibited the best linear correlation with T_m, which is the heat-denaturation temperature midpoint of the corresponding acylated complex of Toho-1 E166A/R274N/R276N. The correlation coefficient was 0.947, indicating that a relationship exists between the kinetic parameters and the stability of the intermediates. The results demonstrate a new method for investigating the catalytic properties of enzymes against any substrates, and a new approach to designing enzymes.