The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase.
The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).
Stack
M S
MS
Department of Biochemistry, University of Louisville, Kentucky 40292.
Gray
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D003903:Deuterium / Q000494:pharmacology; D017666:Deuterium Oxide; D018093:Gelatinases; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D003012:Microbial Collagenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism*; D013552:Swine; D013583:Synovial Membrane / Q000201:enzymology*; D013696:Temperature; D014867:Water / Q000494:pharmacology
D014867:Water; D003903:Deuterium; D010434:Pepsin A; D018093:Gelatinases; D003012:Microbial Collagenase; D017666:Deuterium Oxide