Abstract
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.
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Abbreviations
- Ala-397-CPD-Y:
-
CPD-Y with an alanyl residue at position 397
- Arg-397-CPD-Y:
-
CPD-Y with an arginyl residue at position 397
- Bicine:
-
N,N-bis(2-hydroxyethyl)glycine
- CABS-Sepharose:
-
[N-(eaminocaproyl)-p-aminobenzyl]succinyl-Sepharose 4B
- CD:
-
circular dichroism
- CPD-Y:
-
carboxypeptidase Y
- DFP:
-
diisopropylphosphofluoridate
- EDTA:
-
ethylenediamine tetraacetic acid, sodium salt
- FA:
-
furylacryloyl
- GYBS-Sepharose:
-
[(N-glycyl-L-tyrosine)-p-azo-benzyl]succinyl-Sepharose 4B
- Hepes:
-
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid
- HPLC:
-
high performance liquid chromatography
- Mes:
-
2(N-morpholinolethanesulfonic acid
- SDS:
-
sodium dodecylsulfate. Abbreviations of amino acids and peptides are according to the guidelines of IUPAC-IUB Commission on Biochemical Nomenclature
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Bech, L.M., Breddam, K. Inactivation of carboxypeptidase Y by mutational removal of the putative essential histidyl residue. Carlsberg Res. Commun. 54, 165–171 (1989). https://doi.org/10.1007/BF02904470
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DOI: https://doi.org/10.1007/BF02904470