Abstract
Most methods for studying the kinetic properties of an enzyme involve the determination of initial velocities. When the reaction progress curve shows significant curvature due to depletion of the substrate, accumulation of inhibitory products or instability of the enzyme, estimation of the initial velocity is a subjective and inexact process. Two methods have been suggested [Cornish-Bowden (1975) Biochem. J. 144, 305-312; Boeker (1982) Biochem J. 203, 117-123] that attempt to eliminate this subjective element. The present study offers a third alternative, which is based on fitting a reparameterized form of the integrated Michaelis-Menten equation to the progress curves by non-linear regression. This method yields estimates and standard errors of the initial velocity and of the time to reach 50% reaction. No prior knowledge of the apparent product concentration at zero time or infinite time is required, since both of these quantities are also estimated from the data. It is shown that this method yields reliable estimates of the initial velocity under a wide range of circumstances, including those where the two previously published methods perform poorly.
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Selected References
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- Boeker E. A. Initial rates. A new plot. Biochem J. 1982 Apr 1;203(1):117–123. doi: 10.1042/bj2030117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cornish-Bowden A. The use of the direct linear plot for determining initial velocities. Biochem J. 1975 Aug;149(2):305–312. doi: 10.1042/bj1490305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duggleby R. G., Morrison J. F. The analysis of progress curves for enzyme-catalysed reactions by non-linear regression. Biochim Biophys Acta. 1977 Apr 12;481(2):297–312. doi: 10.1016/0005-2744(77)90264-9. [DOI] [PubMed] [Google Scholar]
- Duggleby R. G., Morrison J. F. The use of steady-state rate equations to analyse progress curve data. Biochim Biophys Acta. 1979 Jun 6;568(2):357–362. doi: 10.1016/0005-2744(79)90303-6. [DOI] [PubMed] [Google Scholar]
- Duggleby R. G. Regression analysis of nonlinear Arrhenius plots: an empirical model and a computer program. Comput Biol Med. 1984;14(4):447–455. doi: 10.1016/0010-4825(84)90045-3. [DOI] [PubMed] [Google Scholar]
- Porter W. R., Trager W. F. Improved non-parametric statistical methods for the estimation of Michaelis-Menten kinetic parameters by the direct linear plot. Biochem J. 1977 Feb 1;161(2):293–302. doi: 10.1042/bj1610293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Selwyn M. J. A simple test for inactivation of an enzyme during assay. Biochim Biophys Acta. 1965 Jul 29;105(1):193–195. doi: 10.1016/s0926-6593(65)80190-4. [DOI] [PubMed] [Google Scholar]
- Wharton C. W., Szawelski R. J. Half-time analysis of the integrated Michaelis equation. Simulation and use of the half-time plot and its direct linear variant in the analysis of some alpha-chymotrypsin, papain- and fumarase-catalysed reactions. Biochem J. 1982 May 1;203(2):351–360. doi: 10.1042/bj2030351. [DOI] [PMC free article] [PubMed] [Google Scholar]