Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background.
To change the substrate preference of carboxypeptidase Y the putative substrate binding pocket was subjected to random mutagenesis. Based upon the three-dimensional structure of a homologous enzyme from wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216, Ile340 and Cys341 are the amino acid residues of carboxypeptidase Y that constitute S1, the binding pocket for the penultimate amino acid side chain of the substrate. We developed a new and generally applicable mutagenesis strategy to facilitate efficient screening of a large number of mutants with multiple changes in carboxypeptidase Y. The key feature is the elimination of wild type background by introducing a nonsense codon at each target site for subsequent mutagenesis by degenerate oligonucleotides. The entire hypothesized S1 binding pocket and subsets of it were subjected to saturation mutagenesis by this strategy, and screening yielded a number of mutant enzymes which have up to 150 times more activity (kcat/Km) towards CBZ-Lys-Leu-OH than the wild type enzyme. All selected mutants with increased activity have mutations at position 178. Mutagenesis of positions 215 and 216 has virtually no effect on the activity, while mutating positions 340 and 341 generally reduces activity.
Olesen
K
K
Department of Yeast Genetics, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Kielland-Brandt
M C
MC
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D014908:Triticum / Q000201:enzymology
D000596:Amino Acids; D003062:Codon; D002268:Carboxypeptidases; C022633:serine carboxypeptidase