Enzyme kinetics temperature effects

One-step hydroxylation of aromatic nucleus with nitrous oxide (N2O) is among recently discovered organic reactions. A high eflSciency of FeZSM-5 zeolites in this reaction relates to a pronounced biomimetic-type activity of iron complexes stabilized in ZSM-5 matrix. N2O decomposition on these complexes produces particular atomic oj gen form (a-oxygen), whose chemistry is similar to that performed by the active oxygen of enzyme monooxygenases. Room temperature oxidation reactions of a-oxygen as well as the data on the kinetic isotope effect and Moessbauer spectroscopy show FeZSM-5 zeolite to be a successfiil biomimetic model. 

A reaction which follows power-law kinetics generally leads to a single, unique steady state, provided that there are no temperature effects upon the system. However, for certain reactions, such as gas-phase reactions involving competition for surface active sites on a catalyst, or for some enzyme reactions, the design equations may indicate several potential steady-state operating conditions. A reaction for which the rate law includes concentrations in both the numerator and denominator may lead to multiple steady states. The following example (Lynch, 1986) illustrates the multiple steady states... 

Kinetic complexity definition, 43 Klinman s approach, 46 Kinetic isotope effects, 28 for 2,4,6-collidine, 31 a-secondary, 35 and coupled motion, 35, 40 in enzyme-catalyzed reactions, 35 as indicators of quantum tunneling, 70 in multistep enzymatic reactions, 44-45 normal temperature dependence, 37 Northrop notation, 45 Northrop s method of calculation, 55 rule of geometric mean, 36 secondary effects and transition state, 37 semiclassical treatment for hydrogen transfer,... 

Removal of calcium from HRP C has a significant effect not only on enzyme activity and thermal stability, but also on the environment of the heme group. The calcium-depleted enzyme has optical, EPR, and H NMR spectra that are different from those of the native enzyme (211). Temperature dependence studies indicate that the heme iron exists as a thermal admixture of high- and low-spin states. Kinetic measurements at pH 7 show that ki, the rate constant for compound I formation, is only reduced marginally from 1.6 0.1 x 10 to 1.4 x lO M s , whereas k, the rate constant for compound II reduction, is reduced from 8.1 1.6 x 10 to 3.6 x lO M s (reducing substrate p-aminobenzoic acid), 44% of its initial value (211). There can be little doubt that this is the main reason for the loss of enzyme activity on calcium removal. 

The study of temperature effects on the reactivation of an enzyme that has been completely unfolded allows one to distinguish between reactivation (referring to kinetic analysis exclusively) and renaturation, the latter of which would reflect both the refolding transition and the formation of misfolded or aggregated byproducts. 

Since Dr. Goldanskii says he is not familiar with Douzou s work, perhaps I can make some response to the question of Dr. Thomas. Douzou s observations definitely do not illustrate a Goldanskii effect. In my understanding, the essential point from Douzou experiments, focusing on enzyme kinetics at temperatures from above 0°C to substantially below, is that the kinetic parameters change in a continuous way. 

The thermodynamics of these reaction systems have been investigated, resulting in methods to predict the direction of a typical reaction a priori. Furthermore, studies on kinetics, enzyme concentration, pH/temperature effects, mixing, and solvent selection have opened up new perspectives for the understanding, modeling, optimization, and possible large-scale application of such a strategy. 

The Effects of Temperature and pH on Enzyme Kinetics and Enzyme De-activation... 

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. 

Purich, D. L., Contemporary Enzyme Kinetics and Mechanism. New York Academic Press, 1983. Selected chapters from Methods in Enzymology. Detailed information on how to analyze kinetic data and on effects of temperature, pH, and inhibitors. 

The effect of temperature satisfies the Arrhenius relationship where the applicable range is relatively small because of low and high temperature effects. The effect of extreme pH values is related to the nature of enzymatic proteins as polyvalent acids and bases, with acid and basic groups (hydrophilic) concentrated on the outside of the protein. Finally, mechanical forces such as surface tension and shear can affect enzyme activity by disturbing the shape of the enzyme molecules. Since the shape of the active site of the enzyme is constructed to correspond to the shape of the substrate, small alteration in the structure can severely affect enzyme activity. Reactor s stirrer speed, flowrate, and foaming must be controlled to maintain the productivity of the enzyme. Consequently, during experimental investigations of the kinetics enzyme catalyzed reactions, temperature, shear, and pH are carefully controlled the last by use of buffered solutions. 

Various kinetic methods in the enzyme catalysis has been described elsewhere (Likhtenshtein, 1988a Gates, 1991 Bugg, 1997 Comish-Bowden, 1995, 2001 Varfolomeev and Gurevich, 1998) Fersht,1999 Gutfreund,. 1995 Hammes, 2000 Leninger et al.1993 ). In this section we concentrate on recent developments in methods of the kinetic isotope effect, transition state analoges, and nanosecond temperature jump techniques. 

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