Increased hydrophobicity of the S′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis
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Increased hydrophobicity of the S′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis. / Winther, Jakob R.; Kielland-Brandt, Morten C.; Breddam, Klaus.
I: Carlsberg Research Communications, Bind 50, Nr. 5, 1985, s. 273-284.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Increased hydrophobicity of the S′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis
AU - Winther, Jakob R.
AU - Kielland-Brandt, Morten C.
AU - Breddam, Klaus
PY - 1985
Y1 - 1985
N2 - Chemical modification of Met-398 in carboxypeptidase Y from yeast has previously been shown to affect the specificity of the enzyme with respect to the P′1 position. To confirm and further study the role of Met-398, this residue was substituted by a leucyl residue by means of site-directed mutagenesis. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment of PRC1, the structural gene for carboxypeptidase Y, using a dodecanucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of the carboxypeptidase Y gene had been mutated from ATG to TTG while the rest of the subcloned region was conserved. This sequence was then reintroduced into the original PRC1 gene context and a ‡prc1 yeast strain was transformed with the resulting plasmid DNA. The mutant enzyme, Leu-398-carboxypeptidase Y, was isolated by affinity chromatography and shown to have the same molecular weight, N-terminal amino acid sequence and sugar content as carboxypeptidase Y. Cyanogen bromide degradation confirmed the absence of Met-398. Leu-398-CPD-Y was characterized kinetically using a series of N-blocked dipeptide and ester substrates with varying groups in the P′1 position. Compared with the wild type enzyme Leu-398-carboxypeptidase Y showed an increased affinity towards substrates with bulky and hydrophobic groups in the P′1 position. This is consistent with the leucyl side-chain being slightly smaller and more hydrophobic than the methionyl side-chain.
AB - Chemical modification of Met-398 in carboxypeptidase Y from yeast has previously been shown to affect the specificity of the enzyme with respect to the P′1 position. To confirm and further study the role of Met-398, this residue was substituted by a leucyl residue by means of site-directed mutagenesis. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment of PRC1, the structural gene for carboxypeptidase Y, using a dodecanucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of the carboxypeptidase Y gene had been mutated from ATG to TTG while the rest of the subcloned region was conserved. This sequence was then reintroduced into the original PRC1 gene context and a ‡prc1 yeast strain was transformed with the resulting plasmid DNA. The mutant enzyme, Leu-398-carboxypeptidase Y, was isolated by affinity chromatography and shown to have the same molecular weight, N-terminal amino acid sequence and sugar content as carboxypeptidase Y. Cyanogen bromide degradation confirmed the absence of Met-398. Leu-398-CPD-Y was characterized kinetically using a series of N-blocked dipeptide and ester substrates with varying groups in the P′1 position. Compared with the wild type enzyme Leu-398-carboxypeptidase Y showed an increased affinity towards substrates with bulky and hydrophobic groups in the P′1 position. This is consistent with the leucyl side-chain being slightly smaller and more hydrophobic than the methionyl side-chain.
KW - binding sites
KW - Carboxypeptidase Y
KW - chemical modification
KW - kinetics
KW - oligonucleotide
KW - protein engineering
KW - site-directed mutagenesis
KW - substrate specificity
KW - yeast
U2 - 10.1007/BF02907151
DO - 10.1007/BF02907151
M3 - Journal article
AN - SCOPUS:51849182086
VL - 50
SP - 273
EP - 284
JO - Carlsberg Research Communications
JF - Carlsberg Research Communications
SN - 0105-1938
IS - 5
ER -
ID: 200970455