Enzyme catalysis temperature effect

Because both the passive fluctuations and the modulating vibrations can require thermal excitation, this model is capable of accounting for temperature-dependent isotope effects, including those traditionally described by the BeU model. Theoretical studies, which will be the topic of the second and third parts of this three-part series of articles, have not yet produced a consensus on the contribution of specific protein motions to enzyme catalysis. 

Selected entries from Methods in Enzymology [vol, page(s)] Theory, 63, 340-352 measurement, 63, 365 cryosolvent [catalytic effect, 63, 344-346 choice, 63, 341-343 dielectric constant, 63, 354 electrolyte solubility, 63, 355, 356 enzyme stability, 63, 344 pH measurements, 63, 357, 358 preparation, 63, 358-361 viscosity effects, 63, 358] intermediate detection, 63, 349, 350 mixing techniques, 63, 361, 362 rapid reaction techniques, 63, 367-369 temperature control, 63, 363-367 temperature effect on catalysis, 63, 348, 349 temperature effect on enzyme structure, 63, 348. 

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. 

Effects of Temperature and pH on Enzyme-Catalyzed Reactions Detailed Mechanisms of Enzyme Catalysis... 

AT any biochemical processes involve very rapid reactions and transient intermediates. Frequently the rapidity of the reaction causes major technical difficulties in ascertaining the details of the events occurring in the process. One approach to overcome this inherent problem is to utilize the fact that most chemical reactions are temperature dependent. This relationship is quantitatively described by the Arrhenius equation, k = Ae E /RT, where k represents the rate constant, A is a constant (the frequency factor), and Ea is the energy of activation. Consequently, by initiating the reaction at a sufficiently low temperature, interconversion of the intermediates may be effectively stopped and they may be accumulated and stabilized individually. Although the focus of this article is on the application of this low-temperature approach to the study of enzyme catalysis, that is, cryoenzymology, the technique is potentially of much wider biological application (1, 2,3). 

After optimizing the assay conditions, including ionic strength, pH, temperature, activator (Ca ) concentration, and polymer concentration, a calibration curve was developed, which allows the lipid substrate concentration to be determined from the fluorescence intensity. The calibration curve allows the enzyme catalysis kinetics parameters (e.g.. Km and Vmax) to be measured. This PLC turn-off assay is effectively inhibited by known inhibitors (F and EDTA), which demonstrates that the sensor relies on the specific catalysis reaction by PLC. It has been demonstrated to be a sensitive (detection limit 0.5nM enzyme concentration), fast (<5 min), and selective (good specificity over phospholipase A and D, and other nonspecific proteins) PLC assay, which can be carried out at very low initial substrate concentration (in the range of micromolar to nanomolar). 

The solvent d5mamics, i.e., the in vitro and in vivo conditions, and natural breathing , i.e., the quantum fluctuations in the active site, of the enzyme molecule need to be counted in a more complete picture of enzymic catalysis. However, the quantum (fluctuating) nature of the enzymic reactions can be visualized by combining the relationship between the catalytic rate and temperature (7) (DeVault Chance, 1966) with that between the reaction rate and the turnover number or the effective time of reaction (Ar) via Heisenberg relation... 

The rate of catalysis of membrane bound enzymes (Plot B, Figure 1) is more greatly affected than soluble enzymes by lowering the temperature. This is due to the effect of low temperatures on the solidification of the membranes. Thus, an Arrhenius plot of the rate of a membrane-bound enzyme as a function of temperature often shows a discontinuity with a sharp break point (transition temperature) and loss of activity at the temperature where the membrane becomes a gel or more solid phase. 

While it is tempting to explain regulatory and cosolvent effects on the basis of conformational changes favorable or unfavorable to enzyme activity, it is much more difficult to demonstrate the actual involvement, amount, and structural details of such changes. Experimental evidence consists in most cases of bits and pieces provided by techniques such as absorption and fluorescence spectroscopy, circular dichroism, and magnetic circular dichroism. These tools work in solution (and, when desired, at subzero temperatures) to investigate not simply empty enzymes but enzyme—substrate intermediates. However, even with this information, the conformational basis of enzyme activity remains more postulated than demonstrated at the ball and stick level, and in spite of data about the number and sequence of intermediates, definition of their approximate nature, rate constants, and identification of the types of catalysis involved, full explanation of any particular reaction cannot be given and rests on speculative hypothesis. 

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

---