Contribution of the intra- and intermolecular routes in autocatalytic zymogen activation: application to pepsinogen activation

Taking as the starting point a recently suggested reaction scheme for zymogen activation involving intra- and intermolecular routes and the enzyme-zymogen complex, we carry out a complete analysis of the relative contribution of both routes in the process. This analysis suggests the definition of new dimensionless parameters allowing the elaboration, from the values of the rate constants and initial conditions, of the time course of the contribution of the two routes. The procedure mentioned above related to a concrete reaction scheme is extrapolated to any other model of autocatalytic zymogen activation involving intra- and intermolecular routes. Finally, we discuss the contribution of both of the activating routes in pepsinogen activation into pepsin using the values of the kinetic parameters given in the literature.     

Linear mixed irreversible inhibition of the autocatalytic activation of zymogens. Kinetic analysis checked by simulated progress curves

Autocatalytic zymogen activation is a phenomenon of great importance for understanding some fundamental physiological processes involved in the enzyme regulation of gastrointestinal-tract enzymes, blood coagulation, fibrinolysis and the complement system. Examples of such processes are the activation of prekallikrein, trypsinogen and pepsinogen, all of which are controlled by natural proteinase inhibitors. This work studies the kinetics of a general autocatalytic zymogen activation process overlapped by two two-step irreversible inhibitions, i.e. a linear mixed irreversible inhibition. The kinetic equations for the whole course of the reaction are derived for this mechanism. In addition, we determine the corresponding kinetics for a number of particular cases of the general model analyzed, i.e. for reversible and irreversible non-competitive, competitive and uncompetitive inhibition systems which are considered particular cases of the general mechanism studied. The kinetic behavior of the system is related to a parameter, a dimensionless quantity, which shows whether the inhibition or the activation route prevails, in a similar way to that which we have previously carried out for other mechanisms. Finally, based on the kinetic equations obtained, a procedure for discriminating between the different mechanisms considered is suggested. The results of this contribution can be directly applied to most physiological autocatalytic zymogen activations in the presence of an inhibitor, allowing their complete kinetic characterization and suggesting procedures for varying the relative weight of the catalytic and inhibition routes or for changing the predominant route.     

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.     

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.     

Kinetic analysis of a simplified scheme of autocatalytic zymogen activation.

Proteolytic enzymes are usually biosynthesized as somewhat larger inactive precursors known as zymogens. These zymogens must undergo an activation process, usually a limited proteolysis, to attain their catalytic activity. When the activating enzyme and the activated enzyme coincide, the process is an autocatalytic zymogen activation. In the present study, a kinetic analysis of the entire progress curve for the autocatalytic zymogen activation reactions is presented. On the basis of the kinetic equations, a novel procedure is developed to evaluate the kinetic parameters of the reactions. This procedure is particularly useful for the fast zymogen autoactivation reactions. As two examples, the novel procedure is used to analyse the autocatalytic activation of bovine trypsinogen and human blood coagulation factor XII (Hageman factor).     

The kinetics of enzyme systems involving activation of zymogens

A general model of zymogen activation is proposed and explicit kinetic equations for the time courses of the various species and products involved are given. These equations are valid for the whole course of the reaction and therefore for both the transient phase and the steady state. This model is sufficiently general to include mechanisms possessing one or more steps of zymogen activation besides possible steps of inhibition (reversible or irreversible) or inactivation.     

Kinetic analysis of a general model of activation of aspartic proteinase zymogens

Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.     

Kinetic analysis of zymogen autoactivation in the presence of a reversible inhibitor.

Limited proteolysis is a highly specific irreversible process, which can serve to initiate physiological function by converting a precursor protein into a biologically active form. When the activating enzyme and the activated enzyme coincide, the process is an autocatalytic zymogen activation (i.e. reactions in which the zymogens serves as a substrate for the corresponding active enzyme). The activity of proteases is frequently regulated by the binding of specific protease inhibitors. Thus, to understand the biological regulation of proteolysis, one must understand the role of protease inhibitors. In the present study, a detailed kinetic analysis of autocatalytic reaction modulated by a reversible inhibitor is represented. On the basis of the kinetic equation, a novel procedure is developed to evaluate the kinetic parameters of the reaction. As an example of the application of this method, effects of acetamidine, p-amidinobenzamidine and benzamidine on the autoactivation of trypsinogen by trypsin were studied.     

Autocatalytic oscillations in the early phase of the photoreduced methyl viologen-initiated fast kinetic reaction of hydrogenase

The kinetic characteristics of the hydrogen uptake reaction of hydrogenase, obtained by conventional activity measurements, led to the proposal of an autocatalytic reaction step in the hydrogenase cycle or during the activation process. The autocatalytic behavior of an enzyme reaction may result in oscillating concentrations of enzyme intermediates and/or products contributing to the autocatalytic step. This behavior has been investigated in the early phase of the hydrogenase-methyl viologen reaction. To measure fast hydrogenase kinetics, flash-reduced methyl viologen has been used as a light-induced trigger in transient kinetic phenomena associated with intermolecular electron transfer to hydrogenase. Here we report fast kinetic measurements of the hydrogenase-methyl viologen reaction by use of the excimer laser flash-reduced redox dye. The results are evaluated on the assumption of an autocatalytic reaction in the hydrogenase kinetic cycle. The kinetic constants of the autocatalytic reaction, i.e. the methyl viologen binding to and release from hydrogenase, were determined, and limits of the kinetic constants relating to the intramolecular (intraenzyme) reactions were set.     

Kinetics of an autocatalytic zymogen reaction in the presence of an inhibitor coupled to a monitoring reaction

A global kinetic analysis of a model consisting of an autocatalytic zymogen-activation process, in which an irreversible inhibitor competes with the zymogen for the active site of the proteinase, and a monitoring coupled reaction, in which the enzyme acts upon one of its substrates, is presented. This analysis is based on the progress curves of any of the two products released in the monitoring reaction. The general solution is applied to an important particular case in which rapid equilibrium conditions prevail. Finally, we suggest a procedure to predict whether the inhibition or activation route dominates in the steady state of the system. These results generalize our previous analysis of simpler mechanisms.     

Kinetic analysis of a general model of activation of aspartic proteinase zymogens involving a reversible inhibitor. I. Kinetic analysis

Starting from a simple general reaction mechanism of activation of aspartic proteinases zymogens involving a uni- and a bimolecular simultaneous activation route and a reversible inhibition step, the time course equation of the zymogen, inhibitor and activated enzyme concentrations have been derived. Likewise, expressions for the time required for any reaction progress and the corresponding mean activation rates as well as the half-life of the global zymogen activation have been derived. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed.     

Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin

Kinetic analysis of the mechanism of trypsinogen activation by trypsin under rapid equilibrium conditions and certain relationships between the rate constants are presented. The kinetic equations are valid from the beginning of the reaction. In addition, we suggest a procedure, based on the above equations, for the evaluation of the kinetic parameters of the reaction. This procedure is applied to a set of experimental data collected during the activation of bovine trypsinogen by trypsin at 30 degrees C (pH 8.1) in 0.01 M CaCl2. In this system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The apparent rate constant, delta, governing this increase would vary linearly with the trypsinogen concentration, [Z]0, if no Michaelis complex was detectable. However, the increase in delta with [Z]0 is clearly non-linear and fits a hyperbola (delta = k2[Z]0/(Kz + [Z]0)) well.     

Mechanisms and kinetics of procathepsin D activation.

In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.     

Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular autocatalytic mechanism

The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6M urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5-6 mg/l, corresponding to a concentration of at least 80-100nM in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25-0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like alpha2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.     

Investigations on the activation of bovine prochymosin.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.     

Kinetics of a model of autocatalysis, coupling of a reaction in which the enzyme acts on one of its substrates.

A global kinetic analysis is presented of a model of an enzyme autocatalytic process, to which a reaction is coupled, in which the enzyme acts upon one of its substrates. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we determine the corresponding kinetic equations for several particular cases which are characterized by certain relations between the rate constants. Finally, a kinetic data analysis is proposed for one of these particular cases. It can easily be extended to any of the other cases.     

Kinetic Analysis of Self-Replicating Peptides: Possibility of Chiral Amplification in Open Systems

A simplified kinetic model scheme is presented that addresses the main reactions of two recently reported peptide self-replicators. Experimentally observed differences in the autocatalytic efficiency between these two systems-- caused by variations in the peptide sequences--and the possible effect of chiral amplification under heterochiral reaction conditions were evaluated. Our numerical simulations indicated that differences in the catalytic performance are exclusively due to pronounced variations in the rate parameters that control the reversible and hydrophobic interactions in the reaction system but neither to alterations in the underlying reaction network nor to changes in the stoichiometry of the involved aggregation processes. Model predictions further demonstrated the possible existence of chiral amplification if peptide self-replication is performed under heterochiral reaction conditions. Pointing into the direction of a possible cause for biomolecular homochirality, it was found that in open flow reactors, keeping the system under non-equilibrium conditions, a remarkable amplification of enantiomeric excess could be achieved. According to our modeling, this is due to a chiroselective autocatalytic effect and a meso-type separation process both of which are assumed to be intrinsic for the underlying dynamics of heterochiral peptide self-replication.     

Kinetics of the trypsinogen activation by enterokinase and trypsin

A global kinetic analysis of the mechanisms of the trypsinogen activation by enterokinase and trypsin is presented. The kinetic equations of both the transient-phase and the steady-state of these mechanisms are presented. In addition, we here derive the corresponding kinetic equations for the case in which the condition of rapid equilibrium prevails and we propose a kinetic data analysis. The significance of this approach to the treatment of other zymogen activation processes is discussed.     

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.