Search

Substrate specificity studies from this laboratory suggested that Ac-Pro-Leu-Ala-Nva-TrpNH2, and its thioester derivative, Ac-Pro-Leu-Ala-SNva-TrpNH2, would be substrates for stromelysin (SLN). In this paper, we report that both peptides are efficiently hydrolyzed not only by SLN but also by two other matrix metalloproteinases, collagenase and gelatinase, and by the bacterial metalloproteinase thermolysin. The pH-dependence of kc/Km for the SLN-catalyzed hydrolysis of Ac-Pro-Leu-Ala-SNva-TrpNH2 is identical to pH-dependencies for peptide hydrolysis and suggests no major mechanistic differences between thioester and amide hydrolysis by SLN.

The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 μ m and k c K m = 4000 M −1 s −1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with k c K m = 10,700 M −1 s −1 . The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with k c K m = 230,000 M −1 s −1 , by human cathepsin G with k c K m = 4200 M −1 s −1 , and by human plasma plasmin with k c K m = 2800 M −1 s −1 , all with Km much larger than 2 μ m .

Steady-state kinetic parameters were determined at pH 7.4 and 25 °C for the human leukocyte elastase-catalyzed hydrolysis of several N -carbobenzoxy- l -amino acid p -nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that k c and k c K m did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.