Abstract
A general method has been devised for the exact evaluation of the rate constants of the elementary steps of a system consisting of any number of coupled reactions. The method is independent of the structure of the network of coupled steps. The precision of the data evaluation is solely dependent on the quality of the detection unit. The application of the method is illustrated with data collected for the binding of the competitive inhibitor proflavine to chymotrypsin under such conditions that five states of the enzyme are required to interpret the results. In the absence of a substance possessing the binding specificity, the enzyme is present as an equilibrium between an active and an inactive conformer. The latter prevails. The binding of the specific inhibitor releases a slow proton transfer from the medium to the alpha-amino group of Ile-16. Subsequently, the enzyme-inhibitor (or enzyme-substrate) complex re-arranges to the catalytically active form, which is retained until the supply of specific substrate is exhausted. The control features described are general, but are particularly conspicuous under the special environmental conditions used here. A comparison between data for alpha- and delta-forms of chymotrypsin showed that the chain ends of the former impeded the substrate binding and that the activity controlling conformational change occurred in the interior of the enzyme molecule.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Böning W., Havsteen B. H. Characterization of a control switch in chymotrypsin. J Biol Chem. 1982 Jun 25;257(12):6836–6843. [PubMed] [Google Scholar]
- Fersht A. R. Conformational equilibria in -and -chymotrypsin. The energetics and importance of the salt bridge. J Mol Biol. 1972 Mar 14;64(2):497–509. doi: 10.1016/0022-2836(72)90513-x. [DOI] [PubMed] [Google Scholar]
- Garel J. R., Labouesse B. Binding interactions between two ligands and a monomeric protein. Study on indole, protons and chymotrypsin. J Mol Biol. 1970 Jan 14;47(1):41–56. doi: 10.1016/0022-2836(70)90400-6. [DOI] [PubMed] [Google Scholar]
- Havsteen B. H. Linear free energy relationship for osmotic water flow through a membrane. Biophys Chem. 1984 Nov;20(4):305–317. doi: 10.1016/0301-4622(84)80021-6. [DOI] [PubMed] [Google Scholar]
- Havsteen B. H. The kinetics of the two-step interaction of chymotrypsin with proflavin. J Biol Chem. 1967 Feb 25;242(4):769–771. [PubMed] [Google Scholar]
- Hess G. P., McConn J., Ku E., McConkey G. Studies of the activity of chymotrypsin. Philos Trans R Soc Lond B Biol Sci. 1970 Feb 12;257(813):89–104. doi: 10.1098/rstb.1970.0011. [DOI] [PubMed] [Google Scholar]
- Quast U., Engel J., Heumann H., Krause G., Steffen E. Kinetics of the interaction of bovine pancreatic trypsin inhibitor (Kunitz) with alpha-chymotrypsin. Biochemistry. 1974 Jun 4;13(12):2512–2520. doi: 10.1021/bi00709a600. [DOI] [PubMed] [Google Scholar]
- Taylor R. P., Vatz J. B., Lumry R. Control of conformation of -chymotrypsin through chemical modification. Biochemistry. 1973 Jul 17;12(15):2933–2940. doi: 10.1021/bi00739a025. [DOI] [PubMed] [Google Scholar]