Abstract
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 S ′1 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 P ′1 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.
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Abbreviations
- AeMTS:
-
aminoethyl methanethiolsulfonate
- AeS-Cys-CPD-Y:
-
Cys-CPD-Y modified with AeMTS
- Ama-Met-CPD-Y:
-
CPD-Y modified with iodoacetamide
- Arg-CPD-Y:
-
CPD-Y with an arginyl residue at position 398
- BzlMTS:
-
benzyl methanethiolsulfonate
- BzlS-Cys-CPD-Y:
-
Cys-CPD-Y modified with BzlMTS
- Eta-Cys-CPD-Y:
-
Cys-CPD-Y modified with 1-bromo-2-butanone
- Bz:
-
N-benzoyl
- Bzl:
-
benzyl
- CPD-Y:
-
carboxypeptidase Y
- Cys-CPD-Y:
-
CPD-Y with a cysteinyl residue at position 398
- EDTA:
-
ethylene diamine tetraacetic acid, sodium salt
- FA:
-
furylacryloyl
- Hepes:
-
N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid
- HPLC:
-
high performance liquid chromatography
- Mes:
-
2-(N-morpholino) ethane sulfonic acid
- MeMTS:
-
methyl methanethiolsulfonate
- MeS-Cys-CPD-Y:
-
Cys-CPD-Y modified with MeMTS
- Pha-Cys-CPD-Y:
-
Cys-CPD-Y modified with phenacyl bromide
- Pha-Met-CPD-Y:
-
CPD-Y modified with phenacyl bromide
- PrMTS:
-
propyl methanethiolsulfonate
- PrS-Cys-CPD-Y:
-
Cys-CPD-Y modified with PrMTS
- SDS:
-
sodium dodecylsulfate
- Z:
-
N-carbobenzoxy
References
Bech, L.M., J. Nielsen, J.R. Winther, M.C. Kielland-Brandt &K. Breddam: Mutational replacement of methionine by arginine in the S ′1 substrate binding site of yeast carboxypeptidase. Carlsberg Res. Commun. 51, 459–465 (1985)
Breddam, K., F. Widmer &J.T. Johansen: Carboxypeptidase Y catalysed transpeptidations and enzymatic peptide synthesis. Carlsberg Res. Commun. 45, 237–247 (1980)
Breddam, K.: Modification of the single sulfhydryl group in carboxypeptidase Y with mercurials. Influence on enzyme specificity. Carlsberg Res. Commun. 48, 9–19 (1983)
Breddam, K., S. Sørensen &M. Ottesen: Isolation of a carboxypeptidase from malted barley by affinity chromatography. Carlsberg Res. Commun. 48, 217–230 (1983)
Breddam, K. &M. Ottesen: Influence of guanidine derivatives on the specificity of malt carboxypeptidase. Carlsberg Res. Commun 48, 573–582 (1983)
Breddam, K. &M. Ottesen: Malt carboxypeptidase catalysed aminolysis reactions. Carlsberg Res. Commun. 49, 473–481 (1984)
Breddam, K.: Chemically modified carboxypeptidase Y with increased amidase activity. Carlsberg Res. Commun. 49, 535–554 (1984)
Breddam, K.: Modification of amino acid residues in the S ′1 binding site of carboxypeptidase Y. Carlsberg Res. Commun. 49, 627–638 (1984)
Breddam, K. &I. Svendsen: Identification of methionyl and cysteinyl residues in the substrate binding site of carboxypeptidase Y. Carlsberg Res. Commun. 49, 639–645 (1984)
Breddam K. &A. Kanstrup: Cyanylation of the single sulfhydryl group in carboxypeptidase Y with cyanogen bromide. Carlsberg Res. Commun. 52, 65–71 (1987)
Bruice, T.W. &G.L. Kenyon: Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives of L-cysteine. J. Prot. Chem. 1, 47–58 (1982)
Johansen, J.T., K. Breddam &M. Ottesen: Isolation of carboxypeptidase Y by affinity chromatography. Carlsberg Res. Commun. 41, 1–14 (1976)
Kunkel, T.A.: Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492 (1985)
Messing, J.: A multi-purpose cloning system based on the single-stranded DNA bacteriophage Ml3. Recombinant DNA Tech. Bull., NIH publication No. 79–99, 2, 43–48 (1979)
Murray, N.E., W.J. Brammer &K. Murray: Lambdoid phages that simplify recovery of in vitro recombinants. Mol. Gen. Genet. 150, 53–61 (1977)
Riordan, J.F. &B.L. Valle: Reactions with N-ethylmaleimid and p-mercurybenzoate. Meth. Enzymol. 25, 449–456 (1952)
Schechter, I &A. Berger: On the size of the active site of proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157–167 (1967)
Segel, I.: Biochemical calculations. John Wiley & Sons, New York, pp. 393–395 (1968)
Shaked Z, R.P. Szajewski &G.M. Whitesides: Rate of thiol-disulfide interchange reaction involving proteins and kinetic measurements of thiol pKa values. Biochemistry 19, 4156–4166 (1980)
Weber, K., J.R. Pringle &M. Osborne: Measurement of molecular weights by electrophoresis on SDS-acrylamide-gel. Methods Enzymol. 26, 3–27 (1972)
Winther, J.R., M.C. Kielland-Brandt &K. Breddam: Increased hydrophobicity of the S ′1 binding site in carboxypeptidase Y obtained by site-directed mutagenesis. Carlsberg Res. Commun. 50, 273–284 (1985)
Zoller, M.J. &M. Smith: Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Meth. Enzymol. 100, 468–500 (1983)
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Abbreviations of peptides are according to guidelines of IUPAC-IUB Commission on Biochemical Nomenclature. The binding site notation for the enzymes is that ofSchechter andBerger (17). Accordingly, the binding site for the C-terminal amino acid residue is denoted S ′1 and those for the amino acid residues in the amino-terminal direction away from the scissile bond are denoted S1, S2,...,Sn. Amino acids in the substrate are referred to as P1, P2,...,Pn and P ′1 in correspondence with the binding sites.
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Bech, L.M., Breddam, K. Chemical modifications of a cysteinyl residue introduced in the binding site of carboxypeptidase Y by site-directed mutagenesis. Carlsberg Res. Commun. 53, 381–393 (1988). https://doi.org/10.1007/BF02983313
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DOI: https://doi.org/10.1007/BF02983313