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Lecture 8

Lecture 8.docx

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Biological Sciences
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Shelley A.Brunt

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Lecture 8: Mechanisms of Enzyme Action Measurement of K andmV max: Double-reciprocal (lineweaver-Burk) plot to determine V max and K m The MichaelisMenten equation can be rewritten in order to obtain values for Vmax and Km from straight lines on graphs. The most commonly used transformation is the double- reciprocal, or LineweaverBurk, plot in which the values of 1/v0 are plotted against 1/[S] (Figure 5.6 Slide 2). The absolute value of 1/Km is obtained from the intercept of the line at the x axis, and the value of 1/Vmax is obtained from the y intercept. Although double-reciprocal plots are not the most accurate methods for determining kinetic constants, they are easily understood and provide recognizable patterns for the study of enzyme inhibition. Values of kcat can be obtained from measurements of Vmax only when the absolute concentration of the enzyme is known. Values of Km can be determined even when enzymes have not been purified provided that only one enzyme in the impure preparation can catalyze the observed reaction. Kinetics of Multisubstrate reactions Lets consider a reaction in which two substrates, A and B, are converted to products P and Q. E + A + B (EAB) E + P + Q Kinetic measurements for such multisubstrate reactions are a little more complicated than simple one-substrate enzyme kinetics. Can determine K fmr each reaction in the presence of saturating amount of each substrate. Multisubstrate reactions can occur by several different kinetic schemes. These schemes are called kinetic mechanisms because they are derived entirely from kinetic experiments. Sequential reactions require all the substrates to be present before any product is released. Sequential reactions can be either ordered, with an obligatory order for the addition of substrates and release of products, or random with no specific order of binding or release required for the substrate and product. In ping-pong reactions, a product is released before all the substrates are bound. In a bisubstrate ping-pong reaction, the first substrate is bound, the enzyme is altered by substitution, and the first product is released. Then the second substrate is bound, the altered enzyme is restored to its original form, and the second product is released. A ping-pong mechanism is sometimes called a substituted-enzyme mechanism because of the covalent binding of a portion of a substrate to the enzyme. The binding and release of ligands in a ping-pong mechanism are usually indicated by slanted lines. The two forms of the enzyme are represented by E (unsubstituted) and F (substituted). In ping-pong reactions, one substrate is bound and a product is released, leaving a substituted enzyme. A second substrate is then bound and a second product released, restoring the enzyme to its original form.Specific Inhibition of enzyme action An enzyme inhibitor (I) is a compound that binds to an enzyme and interferes with its activity. Inhibitors can act by preventing the formation of the ES complex or by blocking the chemical reaction that leads to the formation of product. As a general rule, inhibitors are small molecules that bind reversibly to the enzyme they inhibit. Some inhibitors bind covalently to enzymes causing irreversible inhibition but most biologically relevant inhibition is reversible. Usually inhibitors are small molecules that bind reversibly to the enzyme, although some bind irreversibly (can be determined if inhibitor can be removed by dialysis or gel filtration chromatography it is reversible). Most biologically relevant inhibitors bind reversibly via noncovalent forces similar to substrate enzyme interactions. There are natural inhibitors in cells and artificial inhibitors used in experiments. Reversible inhibitors are bound to enzymes by the same weak, noncovalent forces that bind substrates and products. The equilibrium between free enzyme (E) plus inhibitor (I) and the EI complex is characterized by a dissociation constant. In this case, the constant is called the inhibition constant, K.i E + I EI
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