2.5 Enzymes

Enzymes, like all positive catalysts, dramatically increase the rate of a given reaction. Enzyme kinetics is principally concerned with the measurement and mathematical description of this reaction rate and its associated constants. For many steps in metabolism, enzyme kinetic properties have been determined, and this information has been collected and organized in publicly available online databases (www.brenda.uni-koeln.de). In the first section of this chapter, we review the fundamentals of enzyme kinetics and provide an overview of the concepts that will help the metabolic modeler make the best use of this resource. The techniques and methods required to determine kinetic constants from purified enzymes have been covered in detail elsewhere and are not discussed here. In the second section, we will describe recent advances in the high throughput, high sensitivity measurement of enzyme activity, detail the methodology, and discuss the use of high throughput techniques for profiling large numbers of samples and providing a first step in the process of identifying potential regulatory candidates.

Enzymes are biological catalysts, in which it is specific to only one type of reaction and to one small group of reactants called substrates. Enzymes take part in the reaction where the catalyst provides an alternative reaction pathway. The effects of temperature, pH and substrate concentration on the enzymatic activity were studied. The double reciprocal method was used in order to find Michaelis constant, Km. Based from the graph of effect of substrate concentration, it shows the bell shaped curve pattern rather than straight linearly line as in the standard curve graph and the ideal condition of the amylase enzyme is obtained at substrate concentration, [S] = 1.5%, in which the enzyme shows the maximum rate of activity at this concentration. For the effect of temperature, the optimum temperature for the enzyme obtained from the graph is at 50℃, which the enzyme activity is at 2.421 x 10-7 mol/min. Lastly, for the effect of pH, the graph showed that the enzyme activity increased from pH 5 until pH 6 and decreased until pH 7 and later increased again until pH 9. Therefore, pH 6 is the most optimum pH for the enzyme activity.

Biochemical Journal, 1994

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.

Journal of Mathematical Chemistry, 2007

This work presents an alternative analysis of the integrated rate equations corresponding to the simple Michaelis-Menten mechanism without product inhibition. The suggested new results are reached under a minimal set of assumptions and include, as a particular case, the classical integrated Michaelis-Menten equation. Experimental designs and a kinetic data analysis are suggested to the estimation of the maximum steady-state rate, V max , the Michaelis-Menten constant, K m , the initial enzyme * Corresponding author. 789 0259-9791/07/1100-0789/0 © 2006 Springer Science+Business Media, Inc. R. Varón et al. / Integrated form of the Michaelis-Menten Equation concentration, [E] 0 , and the catalytic constant, k 2 . The goodness of the analysis is tested with simulated time progress curves obtained by numerical integration.

Biochemical Journal, 2006

Biochimica Et Biophysica Acta-general Subjects, 2010

Complete analysis of single substrate enzyme-catalyzed reactions has required a separate use of two distinct approaches. Steady state approximations are employed to obtain substrate affinity and initial velocity information. Alternatively, first order exponential decay models permit simulation of the time course data for the reactions. Attempts to use integrals of steady state equations to describe reaction time courses have so far met with little success.Here we use equations based on steady state approximations to directly model time course plots.Testing these expressions with the enzyme β-galactosidase, which adheres to classical Michaelis–Menten kinetics, produced a good fit between observed and calculated values.This study indicates that, in addition to providing information on initial kinetic parameters, steady state approximations can be employed to directly model time course kinetics.Integrated forms of the Michaelis–Menten equation have previously been reported in the literature. Here we describe a method to directly apply steady state approximations to time course analysis for predicting product formation and simultaneously obtain multiple kinetic parameters.