Abstract
Abstract Carboxypeptidase Y from bakers' yeast is a diisopropyl phosphorofluoridate (DFP)-sensitive enzyme. The inactivation by [32P]DFP is accompanied by the formation of 1 mole of labeled serine per mole of enzyme. A 15-residue 32P-labeled (*) peptide has been isolated from a peptic digest and shown to have the sequence His-Ile-Ala-Gly-Glu-Ser*-Tyr-Ala-His-Gly-Tyr-Ile-Pro-Val-Phe. The reaction of the serine residue with DFP is blocked by the presence of p-hydroxymercuribenzoate, which also inactivates the enzyme. The single —SH group of the protein may be near the active center of the enzyme, but the —SH group is not available to iodoacetate or iodoacetamide in the absence of denaturants. The sequence around the reactive serine residue and the properties of the —SH group contribute to an active site which is quite different from that of DFP-sensitive proteinases such as trypsin, chymotrypsin, or subtilisin.
Highlights
The reaction of the serine residue with DFP is blocked by the presence of p-hydroxymercuribenzoate, which inactivates the enzyme
The single -SH group of the protein may be near the active center of the enzyme, but the --SH group is not available to iodoacetate or iodoacetamide in the absence of denaturants
The sequence around the reactive serine residue and the properties of the -SH group contribute to an active site which is quite different from that of DFP-sensitive proteinases such as trypsin, chymotrypsin, or subtilisin
Summary
Carboxypeptidase Y from bakers’ yeast is a diisopropyl phosphorofluoridate (DFP)-sensitive enzyme. The reaction of the serine residue with DFP is blocked by the presence of p-hydroxymercuribenzoate, which inactivates the enzyme. The sequence around the reactive serine residue and the properties of the -SH group contribute to an active site which is quite different from that of DFP-sensitive proteinases such as trypsin, chymotrypsin, or subtilisin. Studies on the proteinases in yeast by Hata and Hayashi and associates (l-3) led to the finding of a carboxypeptidasei which can remove most amino acids, including proline, from the COOH termini of proteins and peptides. Carboxypeptidase Y is subject to inhibition by this reagent [2] and possesses the additional properties of inhibition by p-hydroxymercuribenzoate but not by iodoacetic acid, iodoacetamide, or chelating agents [1, 2]. Enzyme with DFP2 and to study the response toward reagents that react with -SH groups
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