Effect of pH on Enzyme Activity

Each enzyme is catalytically active only in a narrow pH range and, as a rule, each has a pH optimum which is often between pH 5.5 and 7.5 (Table 2.11). 

The optimum pH is affected by the type and ionic strength of the buffer used in the assay. The rea- 

In addition the ionization of dissociable substrates as affected by pH can be of importance to 

An enzyme, E, its substrate. A, and the enzyme-substrate complex formed, EA, depending on pH, form the following equilibria  

Which of the charged states of E and EA are involved in catalysis can be determined by following the effect of pH on V and Km. 

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Figure 8-2. Effect of pH on enzyme activity. Consider, for example, a negatively charged enzyme (EH ) that binds a positively charged substrate (SH ). Shown is the proportion (%) of SH+ [ ] and of EH [///] as a function of pH. Only in the cross-hatched area do both the enzyme and the substrate bear an appropriate charge.

Fig. 2. Effects of pH on Enzyme Activity. Symbols ( ), P-mannanase M-I ( ), P-mannanase M-II (A), P-mannanase M-III.

Figure 3-3. Effect of pH on enzyme activity (Courtesy of Graham Fisher, G.B. Fermentation Ind.).

As expected, the calculated values for go through a maximum and decrease on either side. In this case, because of the way the values were chosen, the maximum rate occurs at a pH of 6. If a sufficient number of values for [H" "] are utilized so that several data points are obtained, the lower extremities of the curve can be approximated as straight lines that intersect a horizontal line drawn tangent to the top of the curve. The values of the points on the pH axis can be shown to represent the values of pKj and pK2. Although there are other possible scenarios for interpreting effects of pH on enzyme activity, the approach shown here leads to the conclusion that has been verified by many experimental systems. Enzymes usually function best over a rather narrow range of pH and there is some optimum pH for a particular enzyme. 

Wang and co-workers [24] reported isolating the enzyme which degrades poly(3HB-co-3HV). The degradation of poly(3HB-co-3HV) was found to depend on the extracellular enzymes present in the supernatant. The effect of pH on enzyme activity was evaluated and it was found that the degrading activity of the enzyme increased upon increasing the pH. However, when the temperature was raised, the enzyme activity decreased as did the degradation of poly(3HB-CO-3HV). 

Figure 2.6 The effect of pH on enzyme activity. Enzyme A has a pH optimum of 3-3, enzyme B a pH optimum of 9.0.

Effect of pH on the activity of PNL. The enzyme exhibited maximum activity at pH 9.5 (figure 6). 

Effect of pH on enzyme denaturation Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic charac ter of the amino acid side chains. 

The effect of pH on the activity and stability of soluble and immobilized invertase was determined always at 37°C by mixing the enzyme with buffer solutions at fixed pH. The buffers were prepared according to Bacilla et al. (13). For soluble invertase, the pH values employed were 3.0,3.5,4.0, 4.5, 5.0, 5.5, 6.0, and 6.5, and for immobilized invertase they were 4.5,5.0, 5.5,6.0, and 6.5. The stability against pH was determined by measuring the residual activity of both forms of invertase after 12 min of enzyme-buffer contact. 

Studies of the effect of pH on the activity of olpho-amylases, befo-amylases, and phosphorylase have indicated that imidazol-ium groups are probably important in all three enzymes in addition, carboxyl groups are thought to be necessary for the activity of the amylases, and a-amino groups for the phosphorylase. 

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. 

Figure 5. Effect of heating on enzyme activity (6). Reaction mixtures contained substrate (2 mg.), 0.1M—acetate-buffer, pH—5.0 (2.0 ml.), and 0.1 ml. of an enzyme solution that had previously been held at the temperature shown for 10 min. Glucose equivalent was then estimated by the standard assay procedure described in the text...

Enzyme Studies. (1) Effect of Temperature and pH on the Inactivation of Cellulase. Details of experiments and results on the effects of temperature on the inactivation and adsorption of Tv cellulase, effect of pH on adsorption, activity, and inactivation of To cellulase, effect of enzyme concentration on saccharification of cellulose, and glucose inhibition of saccharification of cellulose have been reported in a separate communication (3). These data show that small enzyme losses (up to 10%) are difficult to detect. Tv cellulase alone does not appear to lose... 

Fig. 4. Effect of dilution on enzyme activity. Samples of purified GA,o C-20 hydroxylase (0.45 mg protein ml ) were 100-fold diluted with Tris-HCl (0.1 M, pH 7) containing BSA, no addition, or glycerol as indicated. After 15 min on ice, the samples were diluted another 10-fold with an incubation mixture as in Fig. 3, adjusting all samples to 2 mg ml BSA and 5% glycerol. Incubation for 5 min at 30°C, extraction and HPLC followed...

To understand the effects of pH on enzyme-catalyzed reactions, a model must be built that can account for both the pH dependence of the catalytically active functional groups in the enzyme, and any ionizable groups in the substrate. We consider the case where the substrate does not ionize, while ionizable groups are present in the free enzyme and enzyme-substrate (ES) complex. The reactive form of the enzyme and the ES complex is the monoionized (EH or EHS) form of a diaddic (EH2)... 

The effects of solution pH on enzyme activity can be particularly informative in defining steps in catalysis that are most affected by interactions with inhibitors. Ionization of different groups on the enzyme can be critical in substrate binding (i.e.,... 

Figure 8.2 The effect of pH on the enzyme lactate dehydrogenase (EC 1.1.1.27). The enzyme shows maximum activity at pH 7.4 (A). When stored in buffer solutions with differing pH values for 1 h before re-assaying at pH 7.4, it shows complete recovery of activity from pH values between 5 and 9 but permanent inactivation outside these limits (B).

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. 

Effect of pH on the ionization of the active site The concentra tion of H+ affects reaction velocity in several ways. First, the cat alytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or unionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (-NH3+). At alkaline pH, this group is deprotonated, and the rate of the reaction, therefore, declines. 

Figure 7.17. Effects of pH on binding of phosphofructokinase (PFK) to myofibrils (open symbols dashed line) and interacting effects of pH and temperature on the self-assembly of PFK, as indexed by residual PFK activity (filled symbols solid lines). Binding of PFK to myofibrils was measured using PFK-con-taining supernatants and myofibrillar preparations from white locomotory muscle of the teleost Paralabrax nebulifer (Roberts et al., 1998). PFK self-assembly was studied using purified enzyme from a mammal (Spermophilus beecheyi) (Hand and Somero, 1983). The percent residual PFK activity after incubation for 60 min under different combinations of pH and temperature provides an estimate of the fraction of PFK that remains as catalytically active tetramers or aggregates of tetramers. (Figure from Somero, 1997.)...

The effects of dilution and pH on enzyme activity must also be considered, as these factors are highly variable in urine samples. Most enzymes are very labile to acid pH and the activity of many can be decreased substantially in concentrated urine... 

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