Abstract
A novel serine carboxypeptidase has been isolated from malted barley by affinity chromatography. The enzyme has been termed malt carboxypeptidase III in correspondence with the nomenclature suggested byMikola to distinguish it from the previously isolated carboxypeptidases I and II. Malt carboxypeptidase III is a monomer with a molecular weight around 48,000, composed of a single peptide chain, two glucosamine residues and 2% neutral sugar. The peptide chain is N-terminally blocked and it contains a single unblocked cysteinyl residue.
Like other serine carboxypeptidases malt carboxypeptidase III exhibits peptidase activity with an acidic pH optimum. However, unlike other such enzymes, the pH optimum for the esterase activity is also acidic. The kinetic analysis showed that this probably is due to deprotonation of an ionizable group with a pKa around 7 which controls the conformation of the, active site. The transition of the active acidic form into the inactive basic form is reversible but time dependent.
Malt carboxypeptidase III exhibits a preference for substrates with Phe, Met, Ile and Val in the P ′1 position and Phe, Leu, Met and Ala in the P1 position. The enzyme is inhibited by diisopropyl phosphorofluoridate, mercuric ions and various organomercuric compounds.
Article PDF
Similar content being viewed by others
Abbreviations
- BS:
-
benzyl succinic acid
- Bz:
-
benzoyl
- Bzl:
-
benzyl
- CABS-Sepharose:
-
[N-(ε-aminocaproyl)-paminobenzyl] succinyl-Sepharose 4B
- CPD-Mm :
-
malt carboxypeptidase III
- DFP:
-
diisopropyl phosphorofluoridate
- E:
-
enzyme
- EDTA:
-
ethylenediamine tetraacetic acid, sodium salt
- Et-Hg-Cl:
-
ethylmercuric chloride
- FA:
-
furylacryloyl
- GYBS-Sepharose:
-
[(N-glycyl-L-tyrosine)-p-azo-benzyl] succinyl-Sepharose 4B
- Me-Hg-I:
-
methylmercuric iodide
- Mes:
-
2-(N-morpholino)ethane sulfonic acid
- Ph-Hg-Cl:
-
phenylmercuric chloride
- p-HMB:
-
parahydroxymercuribenzoate
- SDS:
-
sodium dodecyl sulfate
- Z:
-
carbobenzoxy
References
Breddam, K.: Modification of the single sulfhydryl group of carboxypeptidase Y with mercurials. Influence on enzyme specificity. Carlsberg Res. Commun. 48, 9–19 (1983)
Breddam, K., S. Bech 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 I. Carlsberg Res. Commun. 48, 573–582 (1982)
Breddam, K. &M. Ottesen: Malt carboxypeptidase I catalyzed 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., S. Bech Sørensen &M. Ottesen: Isolation of carboxypeptidase II from malted barley by affinity chromatography. Carlsberg Res. Commun. 50, 199–209 (1985)
Breddam, K.: Enzymatic properties of malt carboxypeptidase II in hydrolysis and aminolysis reactions. Carlsberg Res. Commun. 50, 309–323 (1985)
Breddam, K.: Serine carboxypeptidases. A review. Carlsberg Res. Commun. 51, 83–128 (1986)
Breddam, K. &A. Kanstrup: Cyanylation of the single sulfhydryl group in carboxypeptidase Y with cyanogen bromide. Carlsberg Res. Commun. 52, 65–71 (1987)
Dubois, M., K. A. Gilles, J. K. Hamilton, P. A., Rebers &F. Smith: Colorimetric method for determination of sugar and related substances. Anal. Chem. 38, 350–356 (1956)
Enari, T.-M.: Break-down of proteins during malting and mashing. EBC-Monograph-IV. EBC-symposium on the relationship malt and beer. Helsinki-November 1980
Goodwin, T. W. &R. A. Morton: The spectrophometric determination of tyrosine and tryptophan in proteins. Biochem. J. 40, 628–632 (1946)
Hess, G. P.: Chymotrypsin-chemical properties and catalysis. The Enzymes, 3rd Ed., p. 213–248 (1971)
Hirs, C. H. W.: Determination of cystine, as cysteic acid. Meth. Enzymol. 11, 59–62 (1967)
Hofmann, T.: Penicillocarboxypeptidases S-1 and S-2. Meth. Enzymol. 45, 587–599 (1979)
Johansen, J. T., K. Breddam &M. Ottesen: Isolation of carboxypeptidase Y by affinity chromatography. Carlsberg Res. Commun. 41, 1–14 (1976)
Mikola, L. &J. Mikola: Mobilization of proline in the starchy endosperm of germinating barley grain. Planta 149, 149–154 (1980)
Mikola, L.: Germinating barley grains contain five acid carboxypeptidases with complementary substrate specificities. Biochim. Biophys. Acta 747, 241–252 (1983)
Mikola, L.: Acid carboxypeptidases in grains and leaves of wheat, Triticum aestivum L. Plant Physiol. 81, 823–829 (1986)
Riordan, J. F. &B. L. Vallee: Reactions with N-ethylmaleimide and p-mercuribenzoate. Meth. Enzymol. 25, 449–456 (1972)
Schechter, J. &B. Berger: On the size of the active site of proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157–162 (1967)
Segel, J. H.: Enzyme Kinetics pp. 227–231, Wiley-Interscience, New York (1975)
Sørensen, S. B., K. Breddam &I. Svendsen: Primary structure of carboxypeptidase I from malted barley. Carlsberg Res. Commun. 51, 475–485 (1986)
Sørensen, S. B., I. Svendsen &K. Breddam: Primary structure of carboxypeptidase II from malted barley. Carlsberg Res. Commun. 52, 285–295 (1987)
Weber, K., J. R. Pringle &M. Osbern: Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Meth. Enzymol. 26, 3–27 (1972)
Winther, J. &K. Breddam: The free sulfhydryl group (Cys341) of carboxypeptidase Y: Functional effects of mutational substitutions. Carlsberg Res. Commun. 52, 263–273 (1987)
Author information
Authors and Affiliations
Additional information
Other abbreviations of amino acids, amino acid derivatives and peptides are according to the guidelines of the IUPAC-IUB Commission on Biochemical Nomenclature. The binding site notation for the enzyme is that ofSchechter andBerger (21). Accordingly, the binding site for the C-terminal amino acid residue of the substrate 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,…, S1. The substrate positions are denoted P ′1 , P1, P2,… P1 in correspondence with the binding sites.
Accepted byS.O. Andersen
Rights and permissions
About this article
Cite this article
Breddam, K., Sørensen, S.B. Isolation of carboxypeptidase III from malted barley by affinity chromatography. Carlsberg Res. Commun. 52, 275 (1987). https://doi.org/10.1007/BF02907170
DOI: https://doi.org/10.1007/BF02907170