Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis.
It is demonstrated that site-directed mutagenesis successfully can be combined with chemical modification creating enzyme derivatives with altered properties. A methionyl residue located in the S1' binding site of carboxypeptidase Y was replaced by a cysteinyl residue and the mutant enzyme was isolated and modified with various alkylating and thioalkylating reagents. Treatment of the mutant carboxypeptidase Y with bulky reagents like phenacyl bromide and benzyl methanethiolsulfonate caused a drastic reduction in the activity towards substrates with bulky leaving groups in the P1' position, i.e. -OBzl, -Val-NH2 and amino acids (except -Gly-OH), while substrates with small groups in that position, i.e. -OMe and -NH2, were hydrolysed with increased rates. The presence of a positive charge, in addition to a bulky group, had a further adverse effect on the activity towards substrates with large leaving groups, whereas the activity towards those with small leaving groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsulfonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively.
Bech
L M
LM
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby.
Breddam
K
K
D000577:Amides / Q000378:metabolism; D001665:Binding Sites; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D010452:Peptide Biosynthesis
D000577:Amides; D004952:Esters; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D003545:Cysteine