Kinetics of autocatalytic zymogen activation measured by a coupled reaction: pepsinogen autoactivation

Abstract

A kinetic study was performed of a model for an autocatalytic zymogen activation process involving both intra- and intermolecular routes, to which a chromogenic reaction in which the active enzyme acts upon one of its substrates was coupled to continuously monitor the reaction. Kinetic equations describing the evolution of species involved in the system with time were obtained. These equations are valid for any zymogen autocatalytic activation process under the same initial conditions. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. In addition, a dimensionless distribution coefficient was defined, which shows mathematically whether the intra- or the intermolecular route prevails once the kinetic parameters involved in the system are known. The validity of the results obtained was checked using simulated curves for the species involved. As an example of application of the method, the system is experimentally illustrated by the continuous monitoring of pepsinogen transformation to pepsin.

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. II. Contribution of the uni- and bimolecular activation routes

“…where l is given by Equation I- (14). If now Equation (3) is inserted into Equation (2) and we take into account the expression of [E] a given by Equation I- (19), we have for [E] b :…”

A kinetic study of a ternary cycle between adenine nucleotides

“…The same holds for NADH concentration. This assumption is common practice in enzyme kinetics, where to derive approximate analytical solutions corresponding either to the transient phase or to the steady state of an enzyme reaction, it is usually assumed that the substrate concentration remains approximately constant [21,33–35] and therefore the results obtained are only valid under these conditions. Taking into account that nonrecycling substrates and also NADH (the chromogenic substrate) are continuously consumed in the reaction medium from the outset of the reaction, the equations obtained here will be less accurate as the reaction time progresses.…”

“…This indicates that the AEC values allowed with given k 1 -and k 3 -values are those that fulfill the following condition: Figure 6 shows a three-dimensional plot of Eqn (34) for a fixed S T -value and two different values of AEC, 0.8 (a) and 0.9 (b), which are normal values in a number of tissues and organisms [41]. It can be seen that Kinetics of a ternary cycle there are pairs of k 1 and k 3 values (relatively small values of k 3 ) that are not allowed by fixed AEC values, leading to negative or infinite k 2 -values.The k 3 ⁄ k 1 relationship enabled for a fixed AEC value can easily be derived from Eqn (35), with the following result:…”

“…According to Eqn (36), when AEC 6 0.5, the relationship between the rates of formation and destruction of ATP (k 3 and k 1 ) can take any value; however, for AEC > 0.5, not all values of k 1 and k 3 are allowed, as can be deduced from Eqn (35). It is obvious that a minimum level of ATP regeneration with respect to ATP consumption is necessary to keep a high AEC value, i.e.…”

Steady-State Kinetics of the Autocatalytic Zymogen Activation: A Comparison with the Michaelis-Menten Reaction Mechanism

“…The significance of the IAZA reaction has motivated enzymologists to derive rate equations that describe the progress curves for zymogen activation [13], the steady-state or rapid equilibrium kinetic of zymogen action in the presence of reversible inhibitors [17], competitive and noncompetitive inhibitors [18], as well as complex inter-and intramolecular zymogen activation [15,19,20]. Although rate equations are derived under the presupposition of steady-state kinetics or QSSA, in many of studies of zymogen activation the steady-state kinetics assumption (or QSSA) has not been properly motivated through the traditional method of scaling [21,22], or through more modern methods that have recently been developed [23,24,25,26].…”