PH-activity profile

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

Krieger, N., and Hastings, J. W. (1968). Bioluminescence pH activity profiles of related luciferase fractions. Science 161 586-589. 

Figure 5. pH activity profile of PGE on polygalacturonate. Mcllvaine buffers were used in the pH range from 2.5 to 5,5, Reducing sugars were detected by the neocuproine method. PGE (2 mg/ml) was 80-times diluted and incubated with 500 pi of a 0.25% (w/v) polygalacturonate solution, at SO C. 

There are two catalytically active residues in pepsin Asp-32 and Asp-215. Their ionizations are seen in the pH-activity profile, which has an optimum at pH 2 to 3, and which depends upon the acidic form of a group of pKa 4.5 and the basic form of a group of pKa 1.1.160,161 The pKa values have been assigned from the reactions of irreversible inhibitors that are designed to react specifically with ionized or un-ionized carboxyl groups. Diazo compounds—such as A-diazoacetyl-L-phenylalanine methyl ester, which reacts with un-ionized carboxyls—react specifically with Asp-215 up to pH 5 or so (equation 16.28).162-164 Epoxides, which react specifically with ionized carboxyls, modify Asp-32 (equation 16.29). 

Enzymes require an optimum pH at which their catalytic activity is maximal. The pH-activity profiles of enzymes indicate the pH at which the catalytic sites are in their necessary state of ionization. The optimal pH of an enzyme may be different from that of its normal environment. The action of enzymes in cells may be regulated by variation in the pH of the surrounding medium. 

Three cyanide-degrading nitrilases were recently cloned and purified and their kinetic profiles were evaluated in order to better understand their applicability to cyanide bioremediation. CynD from Bacilluspumilus Cl and DyngD from Pseudomonas stutzeri exhibit fairly broad pH profiles with >50% activity retained across pH 5.2 to pH 8.0 while the CHT (NHase) from Gloeocercospora sorghi exhibited a more alkaline pH activity profile with almost all of its activity retained at pH 8.5, slightly lower thermal tolerance, and quite different metal tolerance compared with the two bacterial enzymes [46]. 

Due to shifts of the pH-activity profiles, pH values often must be adjusted to obtain the optimal activity of the enzyme under investigation (Maurel and Douzou, 1975). When the above requirements are fulfilled, there is always a residual effect of the cosolvent on enzyme activity. In most cases, such an effect is small compared to the effect of lowering temperature. It must be checked that the effect is instantaneous upon addition of the solvent, independent of time, and fully reversible by infinite dilution or dialysis. If these conditions are not met, one should suspect denaturation. 

An interesting observation is that an enzyme may exhibit different pH activity profiles for various neutral substrates. The explanation of this is that the enzyme binds or transforms such various substrates differently. For example. Taka amylase has different pH optima for long chain amyloses and for low molecular mass substrates. Some specific chemical modifications of the side chains of the enzyme may also alter the pH activity profiles. Kobayashi, Miura and Ichisima (1992) modified the lysine amino groups using bifimctional reagent o-phtalaldehyde, and observed a pronounced shift in the pH-dependence of ohgomaltoside hydrolysis. 

Lyophilized enzymes have a pH memory, meaning that the activity of the enzyme in organic solvent parallels its pH-activity profile of the aqueous solution from which it was lyophilized [36, 79-81]. However, very often acidic or basic mixtures within a nonaqueous reaction mixture such as reactant, products, or impurities, can disrupt this delicate protonation state, leading to changes in catalytic activity. To counteract this potential problem, solid-state buffers have been developed to protect the enzyme s protonation state in the nonaqueous environment [53, 82]. These solid-state buffers contain pairs of crystalline solids that can be intercon-... 

That formal similarities occur among the reactions discussed above raises the question of whether the enzymes responsible might be related structurally, mechanistically, and perhaps, evolutionarily. Insofar as the limited information available allows comparison there would seem to be no obvious basis for supposing that a common derivation or mode of action of these enzymes exists. While data on structure are scanty, variability in functional groups is evidenced by the disparate pH-activity profiles and spectra of sensitivity toward inhibitors, for example. As a first approximation, such criteria set the limits for the kinds of groups which are essential for catalytic action. 

It previously has been pointed out that, potentially, PP glucose phosphotransferase and ATP-glucose phosphotransferase activities of glucose-6-phosphatase are the most potent glucose phosphorylating systems which have been characterized for liver (9, 10, 41, 118). Such a conclusion appears to have possible validity principally at and below pH 7 however (see Fig. 9) because of the nature of the pH-activity profiles of the phosphatase-associated phosphotransferase activities. 

Figure 6. pH-activity profile of cellulolytic enzyme activities from Thermoactinomyces sp. under assay conditions. (Q) CM-cellulase, incubation time 10 min (A) Avicelase, incubation time 20 min (O) /3-glu-cosidase, incubation time 30 min. 

The effect of the dissociation of the [3-carboxyl group of Asp77 in double mutated pepsin on the recognition of Lys residue at the Pi position were examined by pH-dependent hydrolysis of two type of peptide substrates containing Phe or Lys at Pi. The major differences in pH activity profiles were the kcJKm values below pH 4. These values for Pi substrates (peptide B and C) decreased as the pH dropped, while there was no significant change for Pi substrate (peptide A). The Km values were pH independent in all cases. These results are different from those of the studies that the existence of Lys or Arg residues at the P4, P3, P2, P3 , P4 , and P5 positions in the substrates influences the Km values with little effect on kcat in porcine pepsin [41] the ionic interactions between the basic residues in the substrate and the side-chain carboxylates in the S4, S3, S2, S3 , S4 , and S5 subsites contribute to substrate affinity. 

Figure 28. pH-activity profiles of wild type A. saitoi 1,2-a-D-mannosidase and mutants. 

A difference in pH activity profile has historically been one of the principal utsuuQiiuiia between uifferem types of hyHiufuuiuasc, especially tcsuCUiuf mid... 

Activity profile. A histidine residue in the active site of aspartate transcarhamoylase is thought to be important in stabilizing the transition state of the bound substrates. Predict the pH dependence of the catalytic rate, assuming that this interaction is essential and dominates the pH-activity profile of the enzyme. 

Fig. 11. Microcalorimetric investigation of pH-activity profile of invertase immobilized on Eupergit C by direct binding (unpublished results)...

An example of such a result is illustrated in Fig. 11 showing the pH-activity profile of invertase immobilized on Eupergit C. The relative activity plotted in the figure is the ratio of the thermometric signal at given pH divided by the maximum value of the thermometric signal observed at the pH optimum. The value of the pH optimum obtained is comparable to the known value for yeast invertase [38]. 

Enzyme reactions are in many cases characterized by a bell-shaped pH/ activity profile ... 

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