Application Note | June 1, 2012
Biochemical Characterization Of An Enzyme By Calculation Of Km And Vmax According To The Model Of Michaelis And Menten
Source: TecanEnzymes play an important role in the organism by catalyzing (accelerating the rate of) and therefore regulating all metabolic reactions. Enzyme molecules bring the reactants into proximity, lower the activation energy, but are not consumed themselves in the reaction. The easiest enzyme mechanism is shown in equation 1. It involves the interaction of a single substrate molecule (S) with a single enzyme molecule (E) to reversibly form an enzyme-substrate complex (ES) and then an irreversible reaction to regenerate the uncomplexed enzyme and a product (P).
Metabolism is regulated either by variation of amount or activity of the enzyme. Protein biosynthesis and proteolytic degradation regulate the amount of enzyme, whereas lack or surplus of substrate or product, presence or absence of essential or inhibitory metabolites, pH-value and ion strength influence enzyme activity. In order to study the reaction velocity of an enzyme depending on substrate concentration, all other factors affecting the rate at which enzymatic reactions proceed must be kept constant. In this way it is possible to investigate the dependence of reaction velocity on substrate concentration by establishing a binding curve, in which the measured signal is proportional to the concentration of enzyme-substrate complex.