RESUMO
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.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Acilação , Substituição de Aminoácidos , Aztreonam/metabolismo , Varredura Diferencial de Calorimetria , Catálise , Ceftazidima/metabolismo , Desenho de Fármacos , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , beta-Lactamases/genéticaRESUMO
The beta-lactamase Toho-1 exhibits a strong tendency to form merohedrally twinned crystals. Here, the crystal quality of Toho-1 was improved by using surface modification to remove a sulfate ion involved in crystal packing. The surface-modified Toho-1 variant (R274N/R276N) was crystallized under similar conditions to those used for wild-type Toho-1. R274N/R276N did not form merohedrally twinned crystals. The crystals diffracted to a significantly higher resolution (approximately 0.97 A) than the wild-type crystals (1.65 A); they belonged to the same space group and had almost identical unit-cell parameters to those of wild-type Toho-1.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutação/genética , beta-Lactamases/química , beta-Lactamases/genética , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Proteínas Mutantes/química , Propriedades de SuperfícieRESUMO
The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.
Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons/química , Ligação de Hidrogênio , Proteínas Ferro-Enxofre/química , Rhodobacter sphaeroides/enzimologia , Sulfolobus/enzimologia , Sequência de Aminoácidos , Cisteína/química , Complexo III da Cadeia de Transporte de Elétrons/genética , Proteínas Ferro-Enxofre/genética , Mutagênese Sítio-Dirigida , Análise Espectral Raman , Tirosina/químicaRESUMO
The hyperthermophilic archaeal sulredoxin from Sulfolobus tokodaii is a water-soluble high-potential Rieske [2Fe-2S] protein with unique pH-dependent redox properties compared with its mesophilic homologues in cytochrome bc1/b6f complexes. The oxidized recombinant sulredoxin has been crystallized by the hanging-drop vapour-diffusion method using 30%(v/v) polyethylene glycol 400, 0.1 M cadmium chloride and 0.1 M sodium acetate pH 4.6. The crystals diffracted to beyond 2.0 A resolution and belong to the cubic space group F4(1)32, with unit-cell parameter a = 163.00 +/- 0.05 A. The asymmetric unit contains one sulredoxin molecule. Three-wavelength MAD data were collected.
