Temperature, effect enzymic reactions

Understand the effects of pH, ionic strength, and temperature on enzyme reactions. 

FIGURE 14.12 The effect of temperature on enzyme activity. The relative activity of an enzymatic reaction as a fnncdon of tempera-tnre. The decrease in the activity above 50 C is dne to thermal denatnradon. 

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... 

FIGURE 5.7. Effect of changing the cosubstrate and the pH on the kinetics of an homogeneous redox enzyme reaction as exemplified by the electrochemical oxidation of glucose by glucose oxidase mediated by one-electron redox cosubstrates, ferricinium methanol ( ), + ferricinium carboxylate ( ), and (dimethylammonio)ferricinium ( ). Variation of the rate constant, k3, with pH. Ionic strength, 0.1 M temperature 25°C. Adapted from Figure 3 in reference 11, with permission from the American Chemical Society. 

Figure 8.1 The effect of temperature on enzyme-catalysed reactions. The velocity of a chemical reaction increases with increasing temperature (A) but because of the increasing denaturation of the protein, the proportion of active enzyme falls (B). These two processes result in the characteristic temperature profile of an enzyme (C).

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

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. 

The ratio of the turnover number (i.e., Emax/[Etotai]) to the Xn, value of a substrate in a particular enzyme-catalyzed reaction. When kcat and are the true steady-state parameters, this ratio (or the ratio Emax/T m) is an excellent gauge of the specificity of the enzyme for that substrate. The larger the ratio, the more effective that substrate is used by the enzyme under study. In addition, the effects of a number of mechanistic probes of enzyme action on this ratio (for example, pH effects, isotope effects, temperature effects, the influence of various modifiers, etc.) can provide much information on the catalytic and binding mechanism. See... 

Dixon and Webb provided a useful list of various causes for the shape of the temperature effect seen in enzyme-catalyzed reactions (1) effect on enzyme stability (2) effect on the actual velocity of the reaction (especially on kcat) (3) effect on affinity(ies) of the substrate(s)... 

Figure 8.3 The effect of temperature of an enzyme reaction and the effect of the time-period of the activity measurements on the apparent temperature optimum (after Wiseman, 1975). The index numbers indicate the increase of temperature. It is important to note that in all cases the decrease of the rate of product formation is the consequence of partial inactivations only, i.e. the concentration of substrate must be enough to saturate the enzyme even at time...

Kim and Park subsequently reported that ruthenium pre-catalyst 2 racemizes alcohols within 30 min at room temperature [53]. However, when combined with an enzyme (lipase) in DKR at room temperature, very long reaction times (1.3 to 7 days) were required, in spite of the fact that the enzymatic KR takes only a few hours (Scheme 5.24). Despite these compatibility problems, their results constituted an important improvement, since chemoenzymatic DKR could now be performed at ambient temperature to give high yields, which enables non-thermostable enzymes to be used. More recently, we communicated a highly efficient metal- and enzyme-catalyzed DKR of alcohols at room temperature (Scheme 5.24) [40, 54]. This is the fastest DKR of alcohols hitherto reported by the combination of transition metal and enzyme catalysts. Racemization was effected by a new class of very... 

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 rate of cell growth is influenced by temperature, pH, composition of medium, rate of air supply, and other factors. In the case that all other conditions are kept constant, the specific growth rate may be affected by the concentration of a certain specific substrate (the limiting substrate). The simplest empirical expression for the effect ofthe substrate concentration on the specific growth rate is the following Monod equation, which is similar in form to the Michaelis-Menten equation for enzyme reactions ... 

The enzymic phase is affected by the same factors that affect all enzyme reactions. Variation in the amount of enzyme has the largest effect (Castle and Wheelock 1972). Temperature and pH each affect the enzyme reaction in two ways. As temperature is increased the rate of K-casein cleavage increases until the temperature becomes high enough... 

The rate of urea denaturation was inhibited by a variety of anions known to bind to the enzyme in decreasing order of effectiveness, pyrophosphate, 2 -CMP, phosphate, citrate, tartrate, and sulfate (353). This inhibition was greater at pH 5.6 than 7.3. The binding constants were the same as those estimated by inhibition of the enzymic reaction. As with the pH and temperature effects, the anions had no demonstrable effect on the rate of renaturation. 

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. 

The explanation of Ql(l effects just presented is rather typical of treatments found in most textbooks, in which a relatively simplified thermodynamic explanation, based on energy distribution patterns, is developed to account for effects of temperature on reaction rates. Such treatments of temperature effects, while correct overall, are abstract and nonmecha-nistic—a necessary property of thermodynamic explanations—and will be seen to be incomplete in important ways. In particular, thermodynamic treatments that eschew discussions of underlying mechanisms are unable to provide an explicit account of what steps in an enzyme-catalyzed reaction are rate limiting and, thus, responsible for Qio effects. 

The most common enzymatic reactions are those with two or more substrates and as many products. But many of the simpler single-substrate schemes are valuable for the development of kinetic ideas concerning effects of pH, temperature, etc., on enzyme reaction rates. Although the mechanisms of multisubstrate reactions are complicated, their kinetics can often be described by an equation of the form ... 

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