RESUMEN
The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.
Asunto(s)
Aminopeptidasas/metabolismo , Ácido Aspártico/fisiología , Tirosina/fisiología , Secuencia de Aminoácidos , Aminopeptidasas/química , Aminopeptidasas/genética , Catálisis , Dicroismo Circular , Geobacillus stearothermophilus/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Desnaturalización Proteica , Alineación de SecuenciaRESUMEN
Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.
Asunto(s)
Geobacillus stearothermophilus/enzimología , Peróxido de Hidrógeno/química , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/genética , Metionina/química , Sustitución de Aminoácidos/genética , Estabilidad de Enzimas , Leucil Aminopeptidasa/metabolismo , Metionina/genética , Mutagénesis Sitio-Dirigida/genética , Oxidación-ReducciónRESUMEN
A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant gamma-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6-8 and 40 degrees C, respectively. The chloride salt of metal ions Mg(2+), K(+), and Na(+) can activate BLrGGT, whereas that of Pb(2+) dramatically inhibited it. The substrate specificity study showed that L-gamma-glutamyl-p-nitroanilide (L-gamma-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k (cat) of 105 s(-1) and a K (m) of 21 microM when using L-gamma-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important gamma-glutamyl compounds.
Asunto(s)
Bacillus/enzimología , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/metabolismo , Secuencia de Aminoácidos , Bacillus/genética , Bacillus/aislamiento & purificación , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Glutamina/análogos & derivados , Glutamina/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Plomo/farmacología , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Peso Molecular , Cloruro de Potasio/farmacología , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Cloruro de Sodio/farmacología , Especificidad por Sustrato , Temperatura , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/aislamiento & purificaciónRESUMEN
Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70 degrees C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k(cat) value.