KINETIC CONSTANTS FOR THE HYDROLYSIS OF AGGRECAN BY THE PAPAYA PROTEINASES AND THEIR RELEVANCE FOR CHEMONUCLEOLYSIS

The four known proteinases from papaya latex, namely papain (EC 3.4.22 .2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl en dopeptidase (EC 3.4.22.25), were purified to homogeneity and fully cha racterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kineti c constants for aggrecan hydrolysis by the papaya proteinases. The dis appearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the K-m was much larger than 0.5 mu M an d that k(cat)/K-m = 1.2 +/- 0.1 x 10(6) M(-1) s(-1) for chymopapain, 1 .20 +/- 0.08 x 10(6) M-(1) s-(1) for caricain, 0.90 +/- 0.02 x 10(6) M (-1) s(-)1 for papain, and 0.120 +/- 0.005 x 10(6) M(-1) s(-1) for gly cyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggre can hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observati ons, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis. (C) 1995 Academic Pres s, Inc.