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Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A.
- Source :
-
Biochemistry [Biochemistry] 1990 Aug 07; Vol. 29 (31), pp. 7303-9. - Publication Year :
- 1990
-
Abstract
- Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)
- Subjects :
- Amino Acid Sequence
Animals
Antibodies, Monoclonal immunology
Carboxypeptidases immunology
Carboxypeptidases isolation & purification
Carboxypeptidases A
Catalysis
Cattle
Chromatography, Affinity
Epitopes immunology
Esters metabolism
Hydrogen Bonding
Isoenzymes metabolism
Kinetics
Models, Molecular
Molecular Sequence Data
Peptides metabolism
Protein Conformation
Substrate Specificity
Carboxypeptidases metabolism
Saccharomyces cerevisiae Proteins
Subtilisins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 29
- Issue :
- 31
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 1698455
- Full Text :
- https://doi.org/10.1021/bi00483a021