Abstract
The spontaneous and pepsin-catalyzed activation of pepsinogen has been observed and analyzed kinetically. At appropriate protein concentrations (1 mg per ml or less), a kinetically first order reaction was observed in the pH range 1 to 3, implying an intramolecular activation mechanism. Substantiation of the first order reaction came from a linear plot of log pepsinogen concentration versus time, with an ordinate intercept at the log of the initial pepsinogen concentration. Moreover a 10-fold dilution in protein concentration did not diminish the half-life of the activation reaction. The first order rate constant of 4.7 per min at 28° was comparable to the kcat values observed for peptic hydrolysis of synthetic peptide substrates. At pH 4, we observed kinetics consistent with a mixed reaction mechanism involving the bimolecular reaction of a pepsin molecule and a pepsinogen molecule as well as an intramolecular activation reaction. Below pH 3, this second order process was studied by observing the initial rate of pepsinogen activation in the presence of pepsin. When the pH profiles of the first and second order rate constants were compared, the first order rate constant declined much more rapidly as pH increased than did the second order rate constant. This fact explains the change from a predominantly intermolecular activation mechanism at pH 4 to an intramolecular activation below pH 3.
Highlights
At appropriate protein concentrations (1 mg per ml or less), a kinetically first order reaction was observed in the pH range
Substantiation of the first order reaction came from a linear plot of log pepsinogen concentration versus time, with an ordinate intercept at the log of the initial pepsinogen concentration
A IO-fold dilution in protein concentration did not diminish the half-life of the activation reaction
Summary
The spontaneous and pepsin-catalyzed activation of pepsinogen has been observed and analyzed kinetically. At pH 4, we observed kinetics consistent with a mixed reaction mechanism involving the bimolecular reaction of a pepsin molecule and a pepsinogen molecule as well as an intramolecular activation reaction. PH 3, this second order process was studied by observing the initial rate of pepsinogen activation in the presence of pepsin. Herriott characterized the kinetic data below pH 4.0 as due to a mixed reaction, but he proved that autocatalysis was possible below pH 4.0 because the addition of pepsin to pepsinogen accelerated the activation rate (9). A zymogen molecule could catalyze the activation of another zymogen molecule, a second order kinetic process which is distinct from the autocatalytic process Both prorennin and trypsinogen may be capable of this type of activation mechanism (4, 12). An intramolecular activation mechanism, a kinetically first order process, is conceivable and has been proposed for pepsinogen (13). Our study of the pH dependence of the rate constants for intramolecular and autocatalytic activation provides insight into the reasons for a change of predominant mechanism as pT1 is changed
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