Preparation and activation of a spin-labeled pepsinogen.
gen derivative has been prepared, using 3-[[2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrolyl-1-oxy with 3 spin-labels/protein molecule. Two are located in the peptide that is released first (1-16) and the other is in the secondarily removed sequence (17-44). Activation of the labeled zymogen is a faithful model of native pepsinogen activation because the two processes are closely related in rate, pH dependence of rate, and sites of peptide bond cleavage. The ESR signal associated with the bound label changes during activation due to release of the activation peptide. The rate of release (0.5 min(-1) at pH 2, 22 degrees C) is an order of magnitude slower than is the rate of activation, i.e., cleavage of the peptide bond that holds the peptide to the enzyme. Activation in the presence of pepstatin, however, results in peptide release (2 min(-1) at pH 2, 22 degrees C) nearly as fast as activation occurs. At pH values between 2.5 and 3, there is a lag in the change of the ESR signal following acidification. This indicates accumulation of an intermediate with pH 8.5 labile activity that still has its activation peptide attached. The strong temperature dependence of the rate of activation (26 kcal/mol) is reflected neither in reported characteristics of pepsin catalysis nor in the measured rate of release of the activation peptide from the enzyme (13 kcal/mol).
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D007700:Kinetics; D010435:Pepsinogens / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013113:Spin Labels / Q000494:pharmacology*; D013552:Swine
D010435:Pepsinogens; D010446:Peptide Fragments; D013113:Spin Labels