Data analysis enzyme kinetics

Henderson PJR 1993. Statistical analysis of enzyme kinetic data. Enzyme assays. A practical approach. Eisenthal R, Danson MJ, editors. Oxford Oxford University Press pp. 277-316. 

Selected entries from Methods in Enzymology [vol, page(s)] Dilution of enzyme samples, 63, 10 lipolysis substrate effect, 64, 361, 362 dilution jump kinetic assay, 74, 14-19, 28 dilution method [for dissociation equilibria, 61, 65-96 continuous dilution cuvette, 61, 78-96 data analysis, 61, 74, 75 equations, 61, 70-74 errors, 61, 76-78 experimental procedures, 61, 69, 70 merits, 61, 75, 76 theory, 61, 68, 69... 

Data analysis flow chart, 240, 314-315 data point number requirements, 240, 314 determination of enzyme kinetic parameters multisubstrate, 240, 316-319 single substrate, 240, 314-316 enzyme mechanism testing, 240, 322 evaluation of binding processes, 240, 319321 file transfer protocol site, 240, 312 instructions for use, 240, 312-313. 

Selected entries from Methods in Enzymology [vol, page(s)] Enzyme-substrate complex formation, 64, 53 data analysis, 64, 56, 57 extensions of technique, 64, 57-59 as evidence for occurrence of intermediate, 64, 47-59 kinetic equation, 64, 49-52 limitations, 64, 57-59 mixing procedure, 64, 53-56 reaction condition, 64, 56, 57 termination, 64, 56, 57. 

Kinetic analysis of tyrosinase and calculation of constants will be described using graphical analysis by the Michaelis-Menten equation, Lineweaver-Burk equation, or the direct linear curve. Procedures for preparing these graphs are described below. Alternatively, students may use available computer software to graph data and calculate kinetic constants. Recommended enzyme kinetic computer software packages include Enzyme... 

Most literature on enzyme kinetics is devoted to initial rate data and the analysis of reversible effects on enzyme activity. In many applications and process settings, however, the rate at which the enzyme activity declines is of critical importance. This is especially true when considering its long-term use in continuous reactors. In such situations the economic feasibility of the process may hinge on the useful lifetime of the enzyme biocatalyst. The focus of this section is on the mechanisms and kinetics of loss of enzyme activity. It should also be recognised that the alteration of protein structure is central to the practical manipulation of proteins (e.g. precipitation, affinity and other forms of protein chromatography, and purification in general). 

Comish-Bowden, A. (2002) Statistical analysis of enzyme kinetic data, Oxford University Press, Oxford, p.249-268 ... 

Enzymes are biocatalysts, as such they facilitate rates of biochemical reactions. Some of the important characteristics of enzymes are summarized. Enzyme kinetics is a detailed stepwise study of enzyme catalysis as affected by enzyme concentration, substrate concentrations, and environmental factors such as temperature, pH, and so on. Two general approaches to treat initial rate enzyme kinetics, quasi-equilibrium and steady-state, are discussed. Cleland s nomenclature is presented. Computer search for enzyme data via the Internet and analysis of kinetic data with Leonora are described. 

Notes Reprinted from table 9.1 (p. 156) from Analysis of Enzyme Kinetic Data by Athel Cornish-Bowden (1995) by permission of Oxford University Press. 

The fundamental concept of the transition state stabilization was introduced to Linus Pauling in 1948 who said I think that enzymes are molecules that are complementary in structure to the activated complex of the reactions that they catalyze, that is, the molecular configuration that is intermediate between the reacting substances and the product of the reaction . This concept was widely accepted and used for the interpretation of experimental structural and kinetics data on enzyme catalysis, for the design of new substrates and inhibitors and for chemical mimicking of enzyme reactions. Decisive contributions in this area have been made by structural physical methods, X-ray analysis, in particular, and site-directed mutagenesis. 

As an example to illustrate analysis of kinetic data to characterize the mechanism of a real enzyme, here we apply the general compulsory-order ternary mechanism introduced above to citrate synthase to determine kinetic parameters for several isoforms of this enzyme and to elucidate the mechanisms behind inhibition by products and other species not part of the overall chemical reaction. 

Reversible inhibition can be competitive or non-competitive. Competitive inhibitors bind to the active site and compete with the substrate for binding to the enzyme. However this means that increasing the S concentration will progressively outcompete the inhibitor. Accordingly a Lineweaver—Burk analysis of enzyme kinetic data obtained in the presence or absence of a competitive inhibitor will yield the same Fmax (at infinite S concentration) but the Am in the presence of the inhibitor (A, ) will be higher (poorer binding) than the Am measured in the absence of competitive inhibitor. Knowing the inhibitor concentration [I] one can calculate the A) from the relation ... 

Cleland WW (1979) Statistical analysis of enzyme kinetic data. In Methods in Enzymol-ogy, Vol. 63A, DL Punch, Ed. Academic Press, Orlando, FL. 

This chapter presents a brief summary of the essentials of statistics that are particularly appropriate for handling biochemical data. This is followed by a section on the quantitative analysis of experimental results which deals chiefly with binding processes and enzyme kinetics. The chapter concludes with a brief discussion of methods of sequence analysis and databases, including a description of the FASTA and Needleman and Wunsch algorithms which form the basis of most of the sequence alignment methods currently in use. 

The results of biochemical investigations can only rarely be interpreted without some form of quantitative analysis of the experimental data. In this chapter, we describe methods that can be used for such analysis taking typical biochemical topics such as enzyme kinetics and the thermodynamics and kinetics of molecular interactions as our examples. The aim of the computer-based exercises in this chapter is to provide the reader with direct experience of methods of data analysis that, we hope, will enable them to apply these approaches to their own data. We also indude a short revision of the essentials of thermodynamics and kinetics relevant to the applications discussed. 

Calibration is necessary for in-situ spectrometry in TLC. Either the peak height or the peak area data are measured, and used for calculation. Although the nonlinear calibration curve with an external standard method is used, however, it shows only a small deviation from linearity at small concentrations [94.95 and fulfils the requirement of routine pharmaceutical analysis 96,97J. One problem may be the saturation function of the calibration curve. Several linearisation equations have been constructed, which serve to calculate the point of determination on the basis of the calibration line and these linearisation equations are used in the software of some scanners. A more general problem is the saturation function of the calibration curve. It is a characteristic of a wide variety of adsorption-type phenomena, such as the Langmuir and the Michaelis-Menten law for enzyme kinetics as detailed in the literature [98. Saturation is also evident for the hyperbolic shape of the Kubelka-Munk equation that has to be taken into consideration when a large load is applied and has to be determined. 

CornisH Bowden, a, Analysis of Enzyme Kinetic Data, New York Oxford University Press, 1995. 

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