PH, effect on enzyme reactions

Activity and stability are often comparable to values in aqueous media. Many substrates which cannot be made to react in water or in pure organic solvents such as hexane owing to lack of solubility can be brought to reaction in microemulsions. Whereas enzyme structure and mechanism do not seem to change upon transition from water to the microemulsion phase (Bommarius, 1995), partitioning effects often are very important. Besides an enhanced or diminished concentration of substrates in the vidnity of microemulsion droplets and thus of enzyme molecules, the effective pH values in the water pool of the droplets can be shifted in the presence of charged surfactants. Frequently, observed acceleration or deceleration effects on enzyme reactions can be explained with such partitioning effects (Jobe, 1989). 

Other properties that have to be considered in enzyme reaction engineering are pH effects on the reaction... 

This book starts with a review of the tools and techniques used in kinetic analysis, followed by a short chapter entitled How Do Enzymes Work , embodying the philosophy of the book. Characterization of enzyme activity reversible and irreversible inhibition pH effects on enzyme activity multisubstrate, immobilized, interfadal, and allosteric enzyme kinetics transient phases of enzymatic reactions and enzyme... 

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 major drawback of this reaction system is the high energy and equipment costs due to the use of high pressures. In addition, the use of supercritical carbon dioxide can have adverse effects on enzymes, for example, by decreasing the pH of the microenvironment of the enzyme, by the formation of carbamates owing to covalent modification of free amino groups at the surface of the protein and by deactivation during pressurisation-depressurisation cycles [4]. 

Repeatedly, researchers have found the phenomenon that enzyme molecules seem to remember the aqueous conditions from which they were prepared (Klibanov, 1995). As mentioned above, early results with enzymes in organic solvents demonstrated that enzymes work best in organic solvents if there are lyophilized from aqueous solution at or near the optimum pH value in water. No specific pH effect of organic solvents on enzyme reactions was found the piCa of amino, carboxylic, and phenolic compounds does not differ by more than 0.3 units in the aqueous and lyophilized state (Costantino, 1997). 

Because this kind of role for histidyl residues is present in diverse types of binding sites, two major benefits of alphastat regulation are evident (Somero, 1986). One benefit is that enzymatic activity can be responsive to changes in pH . Shifts in pH during activity or in concert with transition between life stages are able to effectively titrate the activities of enzymes, to ensure that their function occurs in the appropriate direction and at an appropriate level. A second and closely related benefit is the preservation of reversibility in enzymatic reactions, as seen in the pH effects on Km values of LDH. 

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

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 ... 

The pronounced effects of pH on enzyme reactions emphasize the need to control this variable by means of adequate buffer solutions. Enzyme assays should be carried out at the pH of optimal activity, because the pH-activity curve has its minimum slope near this pH, and a small variation in pH will cause a minimal change in enzyme activity. The buffer system must be capable of counteracting the effect of adding the specimen (e.g, serum itself is a powerful buffer) to the assay system, and the effects of acids or bases formed during the reaction (e.g., formation of fatty acids by the action of lipase). Because buffers have their maximimi buffering capacity close to their pK values, whenever possible a buffer system should be chosen with a pK value within IpH unit of the desired pH of the assay (see Chapter 1). Interaction between buffer ions and other components of the assay system (e.g., activating metal ions) may eliminate certain buffers from consideration. 

A new area of research is the control of pH for nonaqueous enzyme reactions using organic pH buffers. - These buffer materials, which are soluble in nonaqueous media, strongly control the effect of pH on the reaction, and are able to override the pH memory of the enzyme before lyophilization or immobilization from an aqueous solution. - Typically, buffer salts employed are oppositely charged (e.g., / -COOH and / -COONa+, where R is hydrophobic in nature). It is the ratio of these two forms that controls the enzymatic rate. The buffers are believed to function by displacing hydrogen atoms of carboxylic acid groups on the surface of the enzyme, . - ... 

Whereas the effect of changing pH on a reaction involving only small molecules can be immediately informative, the interpretation of the effects of pH on enzyme reactions is more complex, since enzymes have many ionising groups and often react by a series of chemical steps with similar rates. 

A pH effect during the reaction occurs if proton shifts are involved, as is the case in most enzymatic reactions. As enzyme activity is strongly dependent on pH, a pH shift induced by the reaction has to be prevented by buffering or titrating the reaction medium. Problems arise especially in immobilized systems where pH gradients occur within the enzyme matrix if the buffering capacity of the medium is too low Is1 62L In cofactor dependent reactions the influence of pH on the stability of NAD(P)+ and NAD(P)H has to be considered[63 . 

The presence of salts in the enzyme-catalyzed reactions favored plastein production proportionally to their concentration. However, the conformation of the protein was changed by the influence of salts [75], Leonil et al. [85] reported that the pH effect on the precipitation of peptides is more marked than the effect of salt. Investigations were carried out into the precipitation of a group of hydrophobic peptides arising from a tryptic casein hydrolysate. 

Isotope effects on enzyme-catalyzed reactions are only rarely fully expressed, since bond-breaking steps are often faster than other steps such as release of reactants. In order to enhance observed isotope effects and obtain information on the degree of rate Umitation of the bond-breaking step, and the location of other rate-limiting steps along the reaction path, the pH can be raised or lowered so that the chemic reaction becomes rate-Umiting (Cook Cleland, 1981 Cook, 1991). Further, the variations of kinetic isotope effects with pH may be used in a profitable way to distinguish between different reaction mechanisms. 

Several aspects affect the extent and character of taste and smell. People differ considerably in sensitivity and appreciation of smell and taste, and there is lack of a common language to describe smell and taste experiences. A hereditary or genetic factor may cause a variation between individual reactions, eg, phenylthiourea causes a bitter taste sensation which may not be perceptible to certain people whose general abiUty to distinguish other tastes is not noticeably impaired (17). The variation of pH in saUva, which acts as a buffer and the charge carrier for the depolarization of the taste cell, may influence the perception of acidity differently in people (15,18). Enzymes in saUva can cause rapid chemical changes in basic food ingredients, such as proteins and carbohydrates, with variable effects on the individual. 

When discussing the role of reaction medium on enzyme enantioselectivity, the potential effects of (i) water activity [5b,13f,32], (ii) enzyme form, and (iii) pH, should... 

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route to ethyl (/ )-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin (Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently reported (Figure 8.15) [94], The reaction conditions were further optimized to operate at 3 m (330 gL ) substrate, pH 7.5 and 27 °C. Under these conditions, 100% conversion and product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6% (based on total protein, 0.1 U mg-1). It is noted that at pH < 6.0 the reaction stalled at <50% conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting material is of low cost and the nitrilase can be effectively expressed in the Pfenex (Pseudomonas) expression system at low cost, introduction of the critical stereogenic center... 

---