title: The catalytic efficiency (kcat/Km) of the class A beta-lactamase Toho-1 correlates with the thermal stability of its catalytic intermediate analog.
abstract: 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-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.
authors.affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, Sayo, Hyogo 679-5148, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Nitanai
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Takuro
authors.initials: T
authors.lastname: Uchiyama
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
authors.forename: Michiyo
authors.initials: M
authors.lastname: Takehira
authors.forename: Katsuhide
authors.initials: K
authors.lastname: Yutani
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuzawa
authors.forename: Masashi
authors.initials: M
authors.lastname: Miyano
mesh_terms: D000215:Acylation; D019943:Amino Acid Substitution; D001398:Aztreonam / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002442:Ceftazidime / Q000378:metabolism; D015195:Drug Design; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D002442:Ceftazidime; D001618:beta-Lactamases; C495977:beta-lactamase Toho-1, E coli; D001398:Aztreonam