The catalytic efficiency (k(cat)/K-m) of the class A beta-lactamase Toho-1 correlates with the thermal stability of its catalytic intermediate analog

作者：Nitanai Yasushi; Shimamura Tatsuro; Uchiyama Takuro; Ishii Yoshikazu; Takehira Michiyo; Yutani Katsuhide; Matsuzawa Hiroshi; Miyano Masashi^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2010, 1804(4): 684-691.

DOI：10.1016/j.bbapap.2009.10.023

• CTX-M-type beta-lactamase
• Toho-1
• Acyl-intermediate
• Thermal stability
• Differential scanning calorimetry
• beta-lactam antibiotics

The extended-spectrum beta-lactamases are associated with antibiotic resistance. Toho-1 R274N/R276N, a Class A beta-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 beta-lactam antibiotics by Toho-l R274N/R276N, and also measured the thermal stability of the accumulated acyl-intermediates of Toho-1E166A/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., 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.

• 出版日期2010-4