Active-site serine mutants of the Streptomyces albus G beta-lactamase.
By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.
Jacob
F
F
Centre d'lngénierie des Protéines, Université de Liège, Belgium.
Joris
B
B
Frère
J M
JM
D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011489:Protein Denaturation; D012694:Serine; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D009841:Oligonucleotides; D012694:Serine; D008623:Mercaptoethanol; D001618:beta-Lactamases