Specificity of pancreatic elastase with tripeptidyl-p-nitroanilide substrates.
1. The kinetic properties of pancreatic elastase (E.C. 3.4.21.11) were investigated with 33 tripeptidyl-p-nitroanilide substrates, and the Km, kcat and kcat/Km values were determined. 2. The individual contributions of the substrate side chains to the kinetic constants were evaluated by regression analysis. As a result of the additivity of the contributions, the kinetic parameters of any substrate constructed from the amino acid investigated can be predicted. 3. Suc-D-Phe-Pro-Ala-pNA was the best substrate, as far as the Km (Michaelis constant) is concerned. The compound was synthesized and assayed. An excellent correlation was observed between its calculated (14 microM) and experimentally determined (15 microM) Km values. The aldehyde derivatives of this substrate is a competitive inhibitor of elastase (Ki = 0.6 mM). 4. The contribution values of the best substrates permitted us to characterize the topography of subsites involved in the formation of the enzyme-substrate complex. This, in turn, led us to the conclusion that the S3-P3 interaction is relatively less important in the binding of good substrates.
D000813:Anilides / Q000378:metabolism*; D000818:Animals; D006868:Hydrolysis; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism*; D012044:Regression Analysis; D013379:Substrate Specificity; D013552:Swine
D000813:Anilides; D009842:Oligopeptides; D010196:Pancreatic Elastase