TY - JOUR

T1 - Mutational replacement of methionine by arginine in the S′1 substrate binding site of yeast carboxypeptidase

AU - Bech, Lene M.

AU - Nielsen, John

AU - Winther, Jakob R.

AU - Kielland-Brandt, Morten C.

AU - Breddam, Klaus

PY - 1986

Y1 - 1986

N2 - Alkylation of Met-398 in the S′1 binding site of carboxypeptidase Y drastically reduces kcat for hydrolysis of peptides, presumably due to introduction of a positively charged sulfonium ion. In the present work a positive charge has been introduced by means of site-directed mutagenesis, exchanging Met-398 with the cationic arginyl residue. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment of PRC1, the structural gene for carboxypeptidase Y, using an oligonucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of PRC1 (ATG) had been changed to AGG, and the mutated sequence was reintroduced into the original PRC1 gene context. The resulting plasmid was used to transform a yeast strain, carrying a deletion at the prc1 locus and the mutant enzyme was isolated by affinity chromatography. The kcat values for the hydrolysis of N-blocked dipeptide substrates with varying groups in the P′1 position were equally low as for the alkylated Met-398 derivatives, consistent with the expected effects of a positive charge in position 398. The kinetic parameters for the hydrolysis of ester and amide substrates were similar to those obtained with the enzyme alkylated with iodoacetamide.

AB - Alkylation of Met-398 in the S′1 binding site of carboxypeptidase Y drastically reduces kcat for hydrolysis of peptides, presumably due to introduction of a positively charged sulfonium ion. In the present work a positive charge has been introduced by means of site-directed mutagenesis, exchanging Met-398 with the cationic arginyl residue. The mutagenesis was carried out in bacteriophage M13 on a subcloned fragment of PRC1, the structural gene for carboxypeptidase Y, using an oligonucleotide containing the desired mutation as primer for secondary strand synthesis in vitro. A clone was identified in which codon 398 of PRC1 (ATG) had been changed to AGG, and the mutated sequence was reintroduced into the original PRC1 gene context. The resulting plasmid was used to transform a yeast strain, carrying a deletion at the prc1 locus and the mutant enzyme was isolated by affinity chromatography. The kcat values for the hydrolysis of N-blocked dipeptide substrates with varying groups in the P′1 position were equally low as for the alkylated Met-398 derivatives, consistent with the expected effects of a positive charge in position 398. The kinetic parameters for the hydrolysis of ester and amide substrates were similar to those obtained with the enzyme alkylated with iodoacetamide.

KW - binding sites

KW - Carboxypeptidase Y

KW - chemical modification

KW - kinetics

KW - protein engineering

KW - site-directed mutagenesis

KW - substrate specificity

U2 - 10.1007/BF02907318

DO - 10.1007/BF02907318

M3 - Journal article

AN - SCOPUS:51849181794

VL - 51

SP - 459

EP - 465

JO - Carlsberg Research Communications

JF - Carlsberg Research Communications

SN - 0105-1938

IS - 6

ER -