Lene M. Bech

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.

Inactivation of carboxypeptidase Y by mutational removal of the putative essential histidyl residue

Carboxypeptidase Y is a serine carboxypeptidase assumed to contain a catalytic triad similar to the serine endopeptidases. On the basis of the homology between various serine carboxypeptidases His-397 is suspected to be part of the catalytic triad. To test this it was exchanged with Ala and Arg by site-directed mutagenesis of the cloned PRC1 gene. The catalytic efficiency of the mutant enzymes were reduced by a factor of 2 · 104 and 7 · 102, respectively, confirming the key role of His-397 in catalysis. Treatment of Ala-397-CPD-Y with Hg++ or CNBr, hence modifying Cys-341 located in the vicinity of the active site abolished the residual activity of the enzyme, indicating an additional involvement of this residue in catalysis.

Introduction of a free cysteinyl residue at position 68 in the subtilisin Savinase, based on homology with proteinase K

Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this ‐SH group a cysteinyl residue has been introduced by site‐directed mutagenesis into the cysteine‐free subtilisin‐like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the ‐SH group is not involved in catalysis.

Studies of Binding Sites in the Subtilisin from Bacillus Lentus by Means of Site Directed Mutagenesis and Kinetic Investigations

The binding site of a proteolytic enzyme may be divided into a number of subsites, each by multiple interactions securing the binding of a single amino acid residue and the proper alignment of the substrate prior to catalysis. The properties of the amino acid residues which constitute a given binding subsite determine which amino acid residue(s) of the substrate may bind and thus, they provide the basis of subsite specificity. The nature of these interactions may be studied by various techniques but it is a prerequisite that the individual subsites are carefully mapped by kinetic investigations using systematic variations of substrate structures. The recent development of highly efficient donor/acceptor pairs for substrates based on intramolecular fluorescence quenching, allowing the use of long peptide substrates spanning the entire binding site, represents a significant improvement in this context.

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

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 ofPRC1, 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 ofPRC1 (ATG) had been changed to AGG, and the mutated sequence was reintroduced into the originalPRC1 gene context. The resulting plasmid was used to transform a yeast strain, carrying a deletion at theprc1 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.