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.
References
Al-Janabi, J., Hartsuck, J.A., and Tang, J. (1972). Kinetics and mechanism of pepsinogen activation. J. Biol. Chem.247, 4628–4632.10.1016/S0021-9258(19)45033-3Search in Google Scholar
Boatright, K.M. and Salvesen, G.S. (2003). Mechanism of caspase activation. Curr. Opin. Cell. Biol.15, 725–731.10.1016/j.ceb.2003.10.009Search in Google Scholar
Chen, J.M., Kukor, Z., Le Marechal, C., Coth, M., Tsakiris, L., Raguenes, C., Ferec, C., and Sahin-Toth, M. (2003). Evolution of trypsinogen activation peptides. Mol. Biol. Evol.20, 1767–1777.10.1093/molbev/msg183Search in Google Scholar
Darvey, I.G. (1977). Transient phase kinetics of enzyme reactions where more than one species of enzyme is present at the start of the reaction. J. Theor. Biol.65, 465–478.10.1016/0022-5193(77)90208-9Search in Google Scholar
Dunn, B.M. and Kay, J. (1985). Design, synthesis and analysis of new synthetic substrates for the aspartic proteinases. Biochem. Soc. Trans.13, 1041–1043.10.1042/bst0131041Search in Google Scholar
Dunn, B.M., Kammermann, B., and McCurry, K.R. (1984). The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences. Anal. Biochem.138, 68–73.10.1016/0003-2697(84)90770-XSearch in Google Scholar
Dunn, B.M., Jiménez, M., Parten, B.F., Valler, M.J., Rolph, C.E., and Kay, J. (1986). A systematic series of synthetic chromophoric substrates for aspartic proteinases. Biochem. J.237, 899–906.10.1042/bj2370899Search in Google Scholar PubMed PubMed Central
Fehlberg, E. and Runge-Kutta, K. (1970). Formeln vierter und niedrigerer Ordnung mit Schrittweiten-Kontrolle und ihre Anwendung auf Wärmeleitungsprobleme. Computing6, 61–71.10.1007/BF02241732Search in Google Scholar
Fersht, A. (2000). Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding, 3rd ed. (New York, USA: W.H. Freeman and Company).Search in Google Scholar
Fruton, J.S. (1976). The mechanism of the catalytic action of pepsin and related acid proteinases. Adv. Enzymol. Relat. Areas Mol. Biol.44, 1–36.10.1002/9780470122891.ch1Search in Google Scholar PubMed
Fuentes, M.E., Varón, R., García-Moreno, M., and Valero, E. (2005). Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme-zymogen complex. FEBS J.272, 85–96.10.1111/j.1432-1033.2004.04400.xSearch in Google Scholar PubMed
García-Moreno, M., Havsteen, B.H., Varón, R., and Rix-Matzen, H. (1991). Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin. Biochim. Biophys. Acta1080, 143–147.10.1016/0167-4838(91)90141-LSearch in Google Scholar
García-Sevilla, F., Garrido del Solo, C., Duggleby, R.G., García-Cánovas, F., Peyró, R., and Varón, R. (2000). Use of a Windows program for simulation of the progress curves of reactants and intermediates involved in enzyme-catalyzed reactions. Biosystems54, 151–164.10.1016/S0303-2647(99)00071-4Search in Google Scholar
Henderson, P.J.F. (1972). A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors. Biochem. J.127, 321–333.10.1042/bj1270321Search in Google Scholar
Kageyama, T. (1988). Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen. Eur. J. Biochem.176, 543–549.10.1111/j.1432-1033.1988.tb14312.xSearch in Google Scholar
Kageyama, T. and Takahashi, K. (1987). Activation mechanism of monkey and porcine pepsinogens A. One-step and stepwise activation pathways and their relation to intramolecular and intermolecular reactions. Eur. J. Biochem.165, 483–490.Search in Google Scholar
Kageyama, T., Ichinose, M., Miki, K., Athauda, S.B., Tanji, M., and Takahashi, K. (1989). Difference of activation processes and structure of activation peptides in human pepsinogens A and progastricsin. J. Biochem.105, 15–22.10.1093/oxfordjournals.jbchem.a122610Search in Google Scholar
Kanost, M.R., Jiang, H., and Yu, X.Q. (2004). Innate immune responses of a lepidopteran insect, Manduca sexta. Immunol. Rev.198, 97–105.10.1111/j.0105-2896.2004.0121.xSearch in Google Scholar
Knowles, J.R., Sharp, H., and Greenwell, P. (1969). The pH dependence of the binding of competitive inhibitors to pepsin. Biochem. J.113, 343–351.10.1042/bj1130343Search in Google Scholar
Lin, X., Wong, R.N.S., and Tang, J. (1989). Synthesis, purification, and active site mutagenesis of recombinant porcine pepsinogen. J. Biol. Chem.264, 4482–4489.10.1016/S0021-9258(18)83769-3Search in Google Scholar
Lin, X., Lin, Y., Koelsch, G., Gustchina, A., Wlodawer, A., and Tang, J. (1992). Enzymic activities of two-chain pepsinogen, two-chain pepsin, and the amino-terminal lobe of pepsinogen. J. Biol. Chem.267, 17257–17263.10.1016/S0021-9258(18)41920-5Search in Google Scholar
Liu, J.H. and Wang, Z.X. (2004). Kinetic analysis of ligand-induced autocatalytic reactions. Biochem. J.379, 697–702.10.1042/bj20031365Search in Google Scholar PubMed PubMed Central
Lluis, F., Roma, J., Suelves, M., Parra, M., Aniorte, G., Gallardo, E., Illa, I., Rodríguez, L., Hughes, S.M., Carmeliet, P., Roig, M., and Muñoz-Cánoves, P. (2001). Urokinase-dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo. Blood97, 1703–1711.10.1182/blood.V97.6.1703Search in Google Scholar
Magklara, A., Mellati, A.A., Wasney, G.A., Little, S.P., Sotiropoulou, G., Becker, G.W., and Diamandis, E.P. (2003). Characterization of the enzymatic activity of human kallikrein 6: autoactivation, substrate specificity, and regulation by inhibitors. Biochem. Biophys. Res. Commun.307, 948–955.10.1016/S0006-291X(03)01271-3Search in Google Scholar
Manjabacas, M.C., Valero, E., García-Moreno, M., García-Cánovas, F., Rodríguez, J.N., and Varón, R. (1992). Kinetic analysis of the control through inhibition of autocatalytic zymogen activation. Biochem. J.282, 583–587.10.1042/bj2820583Search in Google Scholar
Manjabacas, M.C., Valero, E., García-Moreno, M., and Varón, R. (1995). Kinetic analysis of an autocatalytic process coupled to a reversible inhibition. The inhibition of the system trypsinogen-trypsin by p-aminobenzamidine. Biol. Chem. Hoppe-Seyler376, 577–580.Search in Google Scholar
Manjabacas, M.C., Valero, E., García-Moreno, M., Garrido, C., and Varón, R. (1996). Kinetics of an autocatalytic zymogen reaction in the presence of an inhibitor coupled to a monitoring reaction. Bull. Math. Biol.58, 19–41.10.1007/BF02458280Search in Google Scholar
Manjabacas, M.C., Valero, E., Moreno-Conesa, M., García-Moreno, M., Molina-Alarcón, M., and Varón, R. (2002). Linear mixed irreversible inhibition of the autocatalytic activation of zymogens. Kinetic analysis checked by simulated progress curves. Int. J. Biochem. Cell Biol.34, 358–369.10.1016/S1357-2725(01)00135-2Search in Google Scholar
Marin, F., Roldan, V., and Lip, G.Y. (2003). Fibrinolytic function and atrial fibrillation. Thromb. Res.109, 233–240.10.1016/S0049-3848(03)00259-7Search in Google Scholar
Martin, P. (1984). Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence on pH and effect of enzyme phosphorylation level. Biochim. Biophys. Acta791, 28–36.10.1016/0167-4838(84)90277-2Search in Google Scholar
Mathews, J.H. and Fink, K.D. (1999). Ecuaciones diferenciales ordinarias. In: Métodos Numéricos con MATLAB, 3rd ed., I. Capella, ed. (Madrid, Spain: Prentice Hall), pp. 505–509.Search in Google Scholar
McKay, T.R., Bell, S., Tenev, T., Stoll, V., Lopes, R., Lemoine, N.R., and McNeish, I.A. (2003). Procaspase 3 expression in ovarian carcinoma cells increases surviving transcription which can be countered with a dominant-negative mutant, surviving T34A: a combination gene therapy strategy. Oncogene22, 3539–3547.10.1038/sj.onc.1206417Search in Google Scholar PubMed
Nielsen, F.S. and Foltmann, B. (1993). Activation of porcine pepsinogen A. The stability of two non-covalent activation intermediates at pH 8.5. Eur. J. Biochem.217, 137–142.Search in Google Scholar
Pearton, D.J., Nirunsuksiri, W., Rehemtulla, A., Lewis, S.P., Presland, R.B., and Dale, B.A. (2001). Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates. Exp. Dermatol.10, 193–203.10.1034/j.1600-0625.2001.010003193.xSearch in Google Scholar PubMed
Richter, C., Tanaka, T., and Yada, R.Y. (1998). Mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin. Biochem. J.335, 481–490.10.1042/bj3350481Search in Google Scholar PubMed PubMed Central
Richter, C., Tanaka, T., Koseki, T., and Yada, R.Y. (1999). Contribution of a prosegment lysine residue to the function and structure of porcine pepsinogen A and its active form pepsin A. Eur. J. Biochem.261, 746–752.10.1046/j.1432-1327.1999.00329.xSearch in Google Scholar
Salesse, R. and Garnier, J. (1976). Synthetic peptides for chymosin and pepsin assays: pH effect and pepsin independent-determination in mixtures. J. Dairy Sci.59, 1215–1221.10.3168/jds.S0022-0302(76)84349-4Search in Google Scholar
Segel, I.H. (1975). Enzyme Kinetics (New York, USA: John Wiley & Sons).Search in Google Scholar
Shariat-Madar, Z., Mahdi, F., and Schmaier, A.H. (2004). Recombinant prolylcarboxypeptidase activates plasma prekallikrein. Blood103, 4554–4561.10.1182/blood-2003-07-2510Search in Google Scholar
Shi, Y. (2004). Caspase activation: revising the induced proximity model. Cell117, 855–858.10.1016/j.cell.2004.06.007Search in Google Scholar
Spronk, H.M., Govers-Fiemslag, J.W., and ten Cate, H. (2003). The blood coagulation system as a molecular machine. Bioessays25, 1220–1228.10.1002/bies.10360Search in Google Scholar
Tanaka, T. and Yada, R.Y. (1997). Engineered porcine pepsinogen exhibits dominant unimolecular activation. Arch. Biochem. Biophys.340, 355–358.10.1006/abbi.1997.9925Search in Google Scholar
Valero, E., Varón, R., and García-Carmona, F. (2000). Kinetics of a self-amplifying substrate cycle: ADP-ATP cycling assay. Biochem. J.350, 237–243.10.1042/bj3500237Search in Google Scholar
Varón, R., Román, A., García-Canovas, F., and García-Carmona, F. (1986). Transient phase kinetics of activation of human-plasminogen. Bull. Math. Biol.48, 149–166.10.1007/BF02460020Search in Google Scholar
Varón, R., Havsteen, B.H., Vázquez, A., García-Moreno, M., Valero, E., and García-Cánovas, F. (1990). Kinetics of the trypsinogen activation by enterokinase and trypsin. J. Theor. Biol.145, 123–131.10.1016/S0022-5193(05)80538-7Search in Google Scholar
Varón, R., Havsteen, B.H., García-Moreno, M., Vázquez, A., Tudela, J., and García-Cánovas, F.G. (1991). Kinetics of the trypsinogen activation by enterokinase and or trypsin coupling of a reaction in which the trypsin acts on one of its substrates. J. Mol. Catal.66, 409–419.10.1016/0304-5102(91)80032-XSearch in Google Scholar
Varón, R., Havsteen, B.H., García-Moreno, M., and Vázquez, A. (1992). Kinetics of a model of autocatalysis, coupling of a reaction in which the enzyme acts on one of its substrates. J. Theor. Biol.154, 261–270.10.1016/S0022-5193(05)80407-2Search in Google Scholar
Varón, R., Ruíz-Galea, M.M., Garrido-del Solo, C., García-Sevilla, F., García-Moreno, M., García-Canovas, F., and Havsteen, B.H. (1999). Transient phase of enzyme reactions. Time course equations of the strict and the rapid equilibrium conditions and their computerized derivation. Biosystems50, 99–126.10.1016/S0303-2647(98)00095-1Search in Google Scholar
Wang, J.L. and Edelman, G.M. (1971). Fluorescent probes for conformational states of proteins. IV. The pepsinogen-pepsin conversion. J. Biol. Chem.246, 1185–1191.10.1016/S0021-9258(19)76957-9Search in Google Scholar
Worthington Manual (1998). Enzymes and Related Biochemicals. (Freehold, USA: Worthington Biochemical Corporation).Search in Google Scholar
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