Enzyme kinetic analysis

Purification and Enzyme Kinetic Analysis of Variant GST-pi Proteins... 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

Steady-state. In enzyme kinetic analysis, the time interval when the rate of reaction is approximately constant with time. The term is also used to describe the state of a living cell in which the concentrations of many molecules are approximately constant because of a balancing between their rates of synthesis and breakdown. 

The basic principles and definitions used frequently in enzyme kinetic analysis are discussed below. 

In addition to the thermodynamic constraints on the reaction kinetics, a number of assumptions (including quasi-equilibrium binding and quasi-steady state assumptions) are often invoked in computer modeling of enzyme kinetics. Analysis of enzyme kinetics is treated in greater depth in Chapter 4. 

Enzyme inhibition data are often presented as IC50, the concentration of the inhibitor to cause 50 percent inhibition at one chosen substrate concentration Kt, the inhibition constant (dissociation constant from the inhibitor-enzyme complex) determined by enzyme kinetic analysis (e.g., Dixon plot) and /Cin lcl, the time-dependent inhibition constant for mechanism-based inhibitors. IC50 values can be estimated from the study described earlier. A positive inhibition, defined as dose-dependent inhibition, with the inhibited activity lower than 50 percent of that of the negative control, will require further experimentation to define Ki for a better evaluation of in vivo inhibitory potential. Further, a study to determine Klwul may be performed to evaluate if the inhibitor acts via covalent binding to the active site of the enzyme, leading to time-dependent irreversible inhibition. 

This study has important lessons for enzyme kinetic analysis. The use of pH variation and examination of isotope elfects can be a powerful combination to explore the chemistry of enzyme-catalyzed reactions and to dissect the contributions of individual reaction steps to the net steady-state turnover (27). Examination of the effects of pH on each step of the reaction pathway could resolve the contributions of ionizable groups toward ground-state binding energy and transition-state stabilization. The use of isotope effects by transient-state kinetic methods is more limited than in the steady state due to the errors involved in comparing two rate measurements. In the steady state, the ratio method has allowed isotope effects of less than 1% to be measured accurately (8a, 58). By transient-state kinetics, one would require at least a 10-20% change in rate to demonstrate a convincing difference between two rate measurements in most instances. 

A simple way of evaluating the affect of a single nucleotide polymorphism on enzyme function is to perform transient transfection assays in COS-1 cells. Western blot analysis, with anti-UGTl A antibody, is used to determine protein expression levels of the variants, which often correlate with protein stability. Enzyme kinetic analysis is used to investigate the functional impact of amino acid substitutions. 

The basic principles and definitions that are used frequently in enzyme kinetic analysis are also the foim-dation of chemical kinetics in physical chemistry. Three key concepts in this area are as follows. 

To verify that the active PCB and dibenzo-p-dioxin derivatives were interacting with the substrate (rT ) binding site of the enzyme, we investigated the inhibitory mechanism by enzyme kinetic analysis. The results of these experiments are shown in the double reciprocal (Llneweaver-Burk) plot (20) (Fig. 4). A competitive mechanism is strongly suggested by the common intersection point on the vertical axis for the regression lines. The kinetic data derived from this plot for rT are K - 29 nM 70 pmol of... 

Kamal MA, Qu X, Yu Q-S, Tweedie D, Holloway HW, Li Y, Tan Y, Greig NH (2008) Tetrahydrofurobenzofuran cymserine, a potent butyrylcholinesterase inhibitor and experimental Alzheimer drug candidate, enzyme kinetic analysis. J Neural Transm 115 889-898... 

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