Preparation and activation of a spin-labeled pepsinogen

Abstract

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).

Mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin

“…The first is the dissociation of the prosegment from the active enzyme, and the second is the dramatic change in the conformation of the first 13 residues at the N-terminus of the active enzyme. The dissociation of the prosegment has been examined in pig pepsinogen A [84] and chicken pepsinogen A [85] by spin-labelling of Lys residues in the prosegment and monitoring changes in the ESR spectrum upon acidification to pH 2.0. These studies indicate that dissociation of the prosegment in both species of pepsinogen A proceeds by a first-order reaction at a rate which is one order of magnitude slower than the rate of prosegment cleavage.…”

Evolution in the structure and function of aspartic proteases

“…The remainder of the activation peptide (residues 17-44) is then removed, probably by another pepsin molecule. The intramolecular activation mechanism of porcine pepsinogen has been studied kinetically in some detail [69,70]. In the activation of pepsinogen and progastricsin from several species of mammals, the first cleavage sites in the activation peptides appear to differ [71].…”

Activation of porcine pepsinogen A

“…Twining et al, (1981) observed a stepwise release of prosegment peptides whereas Kageyama and Takahashi (1 983) isolated a non-covalent complex between pepsin and the entire prosegment peptide, but the properties of the complexes were not analyzed.…”

“…The former is stable at pH 8.5 whereas the latter rapidly looses activity at pH 8.5. In subequent investigations on the activation of pepsinogen, the reaction mixtures were brought to pH 8.5 and loss of capacity for activation at pH 2 has generally been taken as a measure for the conversion of pepsinogen into pepsin (Al-Janabi et a]., 1972;Sanny et al, 1975: Marciniszyn et a]., 1976Twining et al, 1981;Glick et al, 1989).The activation process is initiated by a pH-dependent change into an active conformation without cleavage of peptide bonds. The reaction then proceeds by limited proteolyses that finally remove 44 amino acid residues (the prowgment) from the N-terminal end of the zymogen peptide chain.…”

“…The former is stable at pH 8.5 whereas the latter rapidly looses activity at pH 8.5. In subequent investigations on the activation of pepsinogen, the reaction mixtures were brought to pH 8.5 and loss of capacity for activation at pH 2 has generally been taken as a measure for the conversion of pepsinogen into pepsin (Al-Janabi et a]., 1972;Sanny et al, 1975: Marciniszyn et a]., 1976Twining et al, 1981;Glick et al, 1989).…”