Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A.
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)
Solomon
B M
BM
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts.
Larsen
K S
KS
Riordan
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002268:Carboxypeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D043422:Carboxypeptidases A; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D000939:Epitopes / Q000276:immunology; D004952:Esters / Q000378:metabolism; D006860:Hydrogen Bonding; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013381:Subtilisins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004952:Esters; D007527:Isoenzymes; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; C019836:CPS1 protein, S cerevisiae; D013381:Subtilisins