Abstract
Carboxypeptidase Y from yeast contains a free sulfhydryl group (Cys341) which is important to the catalytic efficiency and the stability of the enzyme. A number of mutant enzymes have been constructed using a semi-random oligonucleotide directed mutagenesis method. Mutant enzymes, in which Cys341 had been replaced by Ser, Gly, Gln, and Glu, were subjected to kinetic analysis and found to have greatly reduced activities towards a wide range of dipeptide and ester substrates. However, for the Glu341 substituted enzyme the activity towards Bz-Lys-OMe was 4 fold higher than that of the wild type enzyme. The thermal stability as well as pH stabilities of these mutated enzymes were significantly reduced as compared with wild type carboxypeptidase Y, and the enzymes containing His and Lys at position 341 were inactive. The possible functional relation between Cys341 of carboxypeptidase Y and the free cysteinyl group (Cys72) of proteinase K from Tritirachium album Limber is discussed.
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Abbreviations
- Ches:
-
2(N-Cyclohexylamino)ethanesulfonic acid
- CPD-Y:
-
carboxypeptidase Y
- CPD-Y-Hg+ :
-
CPD-Y modified with Hg++ ions
- EDTA:
-
ethylenediamine tetraacetic acid
- FA:
-
furylacryloyl
- Hepes:
-
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- Mes:
-
2-(N-morpholino) ethanesulfonic acid
- RF:
-
replicative form
- SDS:
-
sodium dodecyl sulfate
- ssDNA:
-
single-stranded DNA
- Tris:
-
tris(hydroxymethyl)aminomethane
- Xaa-341-CPD-Y:
-
CPD-Y with amino acids other than cysteine at position 341
- Xaa:
-
Gly, Ser, Gln, Glu, His, Lys, Asp or Asn
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The binding site notation is that ofSchechter andBerger (18). Accordingly, the binding site of the C-terminal amino acid of the substrate is denoted S ′1 and those of the amino acid residues in the amino terminal direction away from the cleaved bond is denoted S1, S2,… Sn. In analogy to this the subtrate residues are denoted: P ′1 and P1, P2,… P3.
Accepted byH. Klenow
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Winther, J.R., Breddam, K. The free sulfhydryl group (Cys341) of carboxypeptidase Y: Functional effects of mutational substitutions. Carlsberg Res. Commun. 52, 263 (1987). https://doi.org/10.1007/BF02907169
DOI: https://doi.org/10.1007/BF02907169