Enzyme kinetics - A modern approach (original) (raw)
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Enzyme Kinetics: Theory and Practice Reaction Rates and Reaction Order
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.
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.
ENZYMES: Catalysis, Kinetics and Mechanisms
N. S. Punekar
Any living being is a reflection of its enzyme arsenal. We are and do what our enzymes permit. Christian de Duve Enzymes are the lead actors in the drama of life. Without these molecular machines the genetic information stored in DNA is worthless. With rising attention to the fashionable fields like molecular biology, genetic engineering, and biotechnology, the techniques to manipulate DNA have occupied center stage. Being popular, many concepts of molecular biology/genetic engineering are now introduced to undergraduates. Unfortunately, this has happened at the cost of other fundamental facets of biology, including enzymology. In the excitement to collate volumes of data for Systems Biology (and the various "Omics" fashions), the beauty and vigor of careful analysisone enzyme at a timeis neglected. It is an intellectual challenge to assay individual enzymes while avoiding complications due to othersan almost forgotten activity in modern biology. Many in the present generation assume that performing one standard assay will tell you everything about that enzyme. While biochemists spent lifetimes on a single native enzyme, the notion today is that one can characterize a mutant in the morning! Over the last three decades devoted enzymologists have become a rare breed. Many Biology teaching programs have expanded in the areas of molecular and cellular biology while they manage with a makeshift enzymology instructor. New students who are attracted to the study of enzymes do exist, but they find themselves in a very bleak teaching environment. Not surprisingly their numbers are dwindling. Reservoirs that are not replenished may soon run dry. Purpose of This Book Genes for enzymes are routinely fished out, cloned, sequenced, mutated, and expressed in a suitable host. Characterizing the mutant enzyme, however, requires a thorough mechanistic studyboth chemical and kinetic. It is thus an exciting time to do enzymology. Hopefully, this book provides enough basic exposure to make this happen.
Enzyme kinetics: the whole picture reveals hidden meanings
FEBS Journal, 2015
The methodology adopted by Michaelis and Menten in 1913 is still routinely used to characterize the catalytic power and selectivity of enzymes. These kinetic measurements must be performed soon after the purified enzyme is mixed with a large excess of substrate. Other time scales and solution compositions are no less physiologically relevant, but fall outside the range of applicability of the classical formalism. Here we show that the complete picture of an enzyme's mode of function is critically obscured by the limited scope of conventional kinetic analysis, even in the simplest case of a single active site without inhibition. This picture is now unveiled in a mathematically closed form that remains valid over the reaction time for all combinations of enzyme/substrate concentrations and rate constants. Algebraic simplicity is maintained in the new formalism when stationary reaction phases are considered. By achieving this century-old objective, the otherwise hidden role of the reversible binding step is revealed and atypical kinetic profiles are explained. Most singular kinetic behaviors are identified in a critical region of conditions that coincide with typical cell conditions. Because it is not covered by the Michaelis-Menten model, the critical region has been missed until now by low-and high-throughput screenings of new drugs. New possibilities are therefore raised for novel and once-promising inhibitors to therapeutically target enzymes.
Investigation On Enzyme and Kinetics
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.
A century of enzyme kinetic analysis, 1913 to 2013
FEBS Letters, 2013
This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function.