Characterization Of Pronase Carboxypeptidase

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology


Pronase is a commercially available mixture of extracellular proteolytic enzymes secreted by the K-1 strain of Streptomyces griseus. In addition to aminopeptidases and a subtilisin, the components include a trypsin, two chymoelastases and a carboxypeptidase, a protease mixture remarkably similar to that produced by the mammalian pancreas. The trypsin and chymoelastases have varying degrees of homology with the corresponding mammalian serine endopeptidases. Earlier studies showed that Pronase carboxypeptidase was a zinc metalloenzyme with peptidase and esterase activities. The present investigation examined the hypothesis that Pronase carboxypeptidase was homologous to mammalian carboxypeptidase. An improved method of isolation which produced higher yields was developed. Studies revealed that the pattern of esterase reactivation with metal replacement in Pronase carboxypeptidase paralleled that seen with mammalian carboxypeptidase A and B. Studies using benzyloxycarbonylglycyl amino acid dipeptide derivatives revealed that the enzyme had activity towards both basic and aliphatic residues but does not cleave proline or acidic residues. This single enzyme thus will hydrolyze all the amino acids susceptible to both pancreatic carboxypeptidases A and B. Active site studies using beta-phenylpropionate and epsilon-aminocaproate as competitive inhibitors showed that the activities for both basic and aliphatic residues resided at a single active site. A polypeptide carboxypeptidase inhibitor purified elsewhere from potatoes showed a high affinity for the Pronase carboxypeptidase with a K(,i) value of 5 x 10('-8)M. Earlier the only carboxypeptidases inhibited by this protein were pancreatic in origin. This confirms further the similarity of Pronase and pancreatic carboxypeptidases and provides an indication of what the natural target of the inhibitor may be. N-Bromoacetyl-N-methyl-L-phenylalanine was used as an active site probe; in contrast to the findings with pancreatic carboxypeptidase it did not react covalently with the active site of the Pronase enzyme. Partial sequence characterization of the Pronase enzyme revealed a 20% homology with mammalian carboxypeptidases A and B. These results confirm the similarity of the microbial and mammalian enzymes and indicate the early association in evolutionary history of serine endopeptidases with zinc carboxypeptidase.


Chemistry, Biochemistry

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