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

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 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).     

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.     

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.     

The concentration of pepsinogen C in human semen and the physiological activation of zymogen in the vagina

The relationship between male infertility and the pepsinogen C content in semen has been investigated. The activation of the seminal pepsinogen C in the vagina has been studied under physiological conditions. Samples of semen from 48 vasectomized males and from 46 males of infertile couples were analyzed for pepsinogen C by radioimmunoassay. No correlation was found between the level of pepsinogen C and seminal characteristics, including sperm concentration, motility, and morphologic features. The mean concentration of pepsinogen C was 42.2 micrograms/ml; the first, second, and third quartile were 18.4, 29.6, and 57.6 micrograms/ml, respectively. No significant difference in the level of pepsinogen C was observed between semen of normal quality, semen of reduced quality, and semen with aspermia. Activation of pepsinogen C occurred within 3 h when semen was incubated at pH below 5.0 at 37 degrees C. Intravaginal activation was investigated in six experiments in which semen from two males was instilled in three females. In four experiments with two couples, post-coital activation was investigated. Pepsin C activity in vaginal fluid was detected an average of 3 h (range 2-5 h) and 5 h (4-7 h) after instillation or ejaculation, respectively. Vaginal pH had then been below 4.5 for approximately 1 h. Pepsin C activity was present in the vagina for more than 24 h thereafter. It is most likely that seminal pepsin C is without influence on the fertilizing spermatozoon. However, pepsin C may exert a local effect in the vagina by degrading seminal proteins, thus preventing an immunogenic response in females.     

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.     

Vacuolar H(+)-ATPases: intra- and intermolecular interactions

V-ATPases in eukaryotes are heteromultimeric, H(+)-transporting proteins. They are localized in a multitude of different membranes and energize many different transport processes. Unique features of V-ATPases are, on the one hand, their ability to regulate enzymatic and ion transporting activity by the reversible dissociation of the catalytic V(1) complex from the membrane bound proton translocating V(0) complex and, on the other hand, their high sensitivity to specific macrolides such as bafilomycin and concanamycin from streptomycetes or archazolid and apicularen from myxomycetes. Both features require distinct intramolecular as well as intermolecular interactions. Here we will summarize our own results together with newer developments in both of these research areas.     

Purification of Japanese monkey prostate acid protease zymogen and its identification as a pepsinogen C-like zymogen

A pepsinogen C-like acid protease zymogen was found in Japanese monkey prostate extract and seminal plasma by means of the double immunodiffusion method using rabbit anti-pepsinogen C antiserum, and was purified from the prostate by a combination of ammonium sulfate fractionation, DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and immunoadsorption to an anti-pepsinogen C column. The zymogen was purified 6,400-fold in a yield of 13.1%. The purified zymogen gave a single band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The zymogen was converted to the active form by acid treatment at pH 2.8 for 4 h with concurrent reduction of the molecular weight from 41,000 to 36,000. By the double immunodiffusion method, prostate pepsinogen C-like acid protease zymogen, pepsinogen C, lung procathepsin D-II, and their active forms gave a single, fused precipitin line in agar plate with anti-pepsinogen C antiserum, which did not react with cathepsin D and pepsinogen A. Furthermore, the optimal pH of 2.5-3.0, the effect of pepstatin on the activity, and the amino acid compositions were also in good agreement among these three zymogens, showing that they are very similar protease zymogens.     

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.     

Contribution of intra- and interhost dynamics to norovirus evolution

Norovirus (NoV) is an emerging RNA virus that has been associated with global epidemics of gastroenteritis. Each global epidemic arises with the emergence of novel antigenic variants. While the majority of NoV infections are mild and self-limiting, in the young, elderly, and immunocompromised, severe and prolonged illness can result. As yet, there is no vaccine or therapeutic treatment to prevent or control infection. In order to design effective control strategies, it is important to understand the mechanisms and source of the new antigenic variants. In this study, we used next-generation sequencing (NGS) technology to investigate genetic diversification in three contexts: the impact of a NoV transmission event on viral diversity and the contribution to diversity of intrahost evolution over both a short period of time (10 days), in accordance with a typical acute NoV infection, and a prolonged period of time (288 days), as observed for NoV chronic infections of immunocompromised individuals. Investigations of the transmission event revealed that minor variants at frequencies as low as 0.01% were successfully transmitted, indicating that transmission is an important source of diversity at the interhost level of NoV evolution. Our results also suggest that chronically infected immunocompromised subjects represent a potential reservoir for the emergence of new viral variants. In contrast, in a typical acute NoV infection, the viral population was highly homogenous and relatively stable. These results indicate that the evolution of NoV occurs through multiple mechanisms.     

Role of glycosylation in corin zymogen activation

The cardiac serine protease corin is the pro-atrial natriuretic peptide convertase. Corin is made as a zymogen, which is activated by proteolytic cleavage. Previous studies showed that recombinant human corin expressed in HEK 293 cells was biologically active, but activated corin fragments were not detectable, making it difficult to study corin activation. In this study, we showed that recombinant rat corin was activated in HEK 293 cells, murine HL-1 cardiomyocytes, and rat neonatal cardiomyocytes. In these cells, activated corin represented a small fraction of the total corin molecules. The activation of recombinant rat corin was inhibited by small molecule trypsin inhibitors but not inhibitors for matrix metalloproteinases or cysteine proteases, suggesting that a trypsin-like protease activated corin in these cells. Glycosidase digestion showed that rat and human corin proteins contained substantial N-glycans but little O-glycans. Treatment of HEK 293 cells expressing rat corin with tunicamycin prevented corin activation and inhibited its pro-atrial natriuretic peptide processing activity. Similar effects of tunicamycin on endogenous corin activity were found in HL-1 cells. Mutations altering the two N-glycosylation sites in the protease domain of rat corin prevented its activation in HEK 293 and HL-1 cells. Our results indicate that N-linked oligosaccharides play an important role in corin activation.     

Contribution of unfolding and intermolecular architecture to fibronectin fiber extensibility

The extracellular matrix contains components with remarkable mechanical properties, including fibronectin (Fn) fibers with extensibilities of >700% strain. We utilized what we consider a novel technique to quantify the extent of molecular unfolding that contributes to Fn fiber extension, and we compared this behavior with stochastic models of Fn fibers with different molecular arrangements. In vitro unfolding as a function of strain was measured by fluorescently labeling cysteines in modules FnIII7 and III15 in artificial Fn fibers. A calibration technique we also consider novel made it possible to demonstrate that 44% of cysteines in these modules were exposed in Fn fibers strained to 421% extension, up from 8% exposure without strain. In silico unfolding was measured by applying a constant strain rate to a fiber represented by a network of wormlike chain springs, each representing an individual Fn molecule. Unfolding rates were calculated with a tension-dependent stochastic model applied to FnIII modules in each molecule. A comparison of these approaches revealed that only a molecular arrangement permitting unequal mechanical loading of Fn molecules recapitulates in vitro unfolding. These data have implications for Fn-dependent mechanotransduction and give insight into how the molecular architecture of natural materials permits such remarkable extensibility.     

Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2

ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three cysteines predicted to be on the extracellular face of ABCG2. Upon mutation of Cys-592 or Cys-608 to alanine (C592A and C608A), ABCG2 migrated as a dimer in SDS-PAGE under non-reducing conditions; however, mutation of Cys-603 to Ala (C603A) caused the transporter to migrate as a single monomeric band. Despite this change, C603A displayed efficient membrane targeting and preserved transport function. Because the transporter migrated as a dimer in SDS-PAGE, when only Cys-603 was present (C592A-C608A), the data suggest that Cys-603 forms a symmetrical intermolecular disulfide bridge in the ABCG2 homodimer that is not essential for protein expression and function. In contrast to C603A, both C592A and C608A displayed impaired membrane targeting and function. Moreover, when only Cys-592 or Cys-608 were present (C592A/C603A and C603A/C608A), the transporter displayed impaired plasma membrane expression and function. The combined mutation (C592A/C608A) partially restored plasma membrane expression; however, although transport of mitoxantrone was almost normal, we observed impairment of BODIPY-prazosin transport. This supports the conclusion that Cys-592 and Cys-608 form an intramolecular disulfide bridge in ABCG2 that is critical for substrate specificity. Finally, mutation of all three cysteines simultaneously resulted in low expression and no measurable function. Altogether, our data are consistent with a scenario in which an inter- and an intramolecular disulfide bridge together are of fundamental importance for the structural and functional integrity of ABCG2.     

A model of intra- and intermolecular interactions of peptide chains]

Results of attempts to determine the code of interaction of amino acids in peptide chains proceeding from their coding nucleotide sequences have been summarized. According to the model suggested the G/C and A/U complementarity of codon roots determines the mutual binding of coded amino acid residues. Structures of analogs of the immunoactive peptide, a fragment of IgG1 (336-370) EPQVY have been constructed on the basis of the model.     

Simultaneous determination of zymogen activation time and zymogen level using chromogenic substrates in blood coagulation analysis

The amidolytic activities of plasma generated by means of thromboplastin and Ca++, on the one hand, and by means of partial thromboplastin, a contact activator and Ca++, on the other hand, were determined using synthetic, chromogenic factor Xa substrates with low affinity for thrombin (CH3SO2-D-Leu-Gly-Arg-pNA and CH3SO2-D-Nleu-Gly-Arg-pNA). In this way, the activation process by splitting off the p-nitroaniline was followed. Besides the summary detection of factor Xa was obtained after addition of hirudin. During preincubation with partial thromboplastin and contact acti (Actin) in Ca++-free medium, an amidolytic activity so far unidentified was generated that renders evaluation of the activation process difficult. In the test system with partial thromboplastin, factor Xa could not be determined and the thrombin-like activity that can be inhibited by hirudin did not correspond to the amount of prothrombin present in plasma. In contrast, activation of factor X and prothrombin by thromboplastin and Ca++ could be followed and the content of the two zymogenes could be detected simultaneously. In general, under optimized reaction conditions, automated systems might be developed that would provide additional diagnostic information about determination of clotting time, on the one hand, and about quantitative determination of zymogen, on the other hand.