Enzyme ionizable groups

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 ... 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

Bell-shaped activity versus pH profiles arise from two separate active-site ionizations, (a) Enzyme activity increases upon deprotonation of (b) Enzyme activity decreases upon deprotonation of A-H. (c) Enzyme activity is maximal in the pH range where one ionizable group is deprotonated (as B ) and the odier group is protonated (as A-H). 

The pH-dependence of the inhibition also indicated that unprotonated castanospermine is a better inhibitor than the protonated form. However, as essential carboxyl groups of the enzyme ionize in the same range of pH as castanospermine (pK, 6.09), it was not possible to estimate the inhibitory potency of protonated castanospermine. 

In the Kohn-Sham Hamiltonian, the SVWN exchange-correlation functional was used. Equation 4.12 was applied to calculate the electron density of folate, dihydrofolate, and NADPH (reduced nicotinamide adenine dinucleotide phosphate) bound to the enzyme— dihydrofolate reductase. For each investigated molecule, the electron density was compared with that of the isolated molecule (i.e., with VcKt = 0). A very strong polarizing effect of the enzyme electric field was seen. The largest deformations of the bound molecule s electron density were localized. The calculations for folate and dihydrofolate helped to rationalize the role of some ionizable groups in the catalytic activity of this enzyme. The results are,... 

Figure 1. Bell-shaped pH-rate profile for an enzyme that has two ionizable groups affecting enzyme activity. The rate is directly proportional to the degree of ionization of the first group (determined by p/Ci) and inversely proportional to the degree of ionization of the second group (governed by p/<2).

Figure 2. Analysis of the pH-dependence of the key parameters influencing the initial rate behavior of an enzyme containing two ionizable groups that determine catalytic activity.

When it is feasible to identify and characterize a single rate process, the effect of pH is more straightforward. This is frequently the case for nonenzymatic reactions obeying simple rate equations, but fast reaction techniques sometimes allow one to examine a single elementary reaction in an enzymic process. In either case, the behavior shown in Fig. 3 typifies the manner in which an observed reaction rate constant depends on the pT a of an ionizable group. 

A vast class of instabilities in enzymatic systems arises from the pH dependence of enzymatic activity. In general, protein molecules contain a number of ionizing groups, such as (—COOH), which can be ionized to give the negatively charged (—COO") ion, and (—NH2), which can add on a proton to give (—NH ). The active enzyme may be represented as in Fig. 4 then addition of acid or base to the active enzyme may be depicted as in Fig. 5, or schematically... 

For many enzymes a plot of Vmax against pH is a bell-shaped curve (Fig. 9-8). The optimum rate is observed at some intermediate pH, which is often, but not always, in the range of pH 6-9. This type of curve can be interpreted most simply by assuming the presence in the active site of two ionizable groups and three forms of the enzyme with different degrees of protonation E, EH, and EH2. 

Chemical studies also support the indicated mechanism. For example, the P-oxoacid intermediate formed in step b of Eq. 13-48 or Fig. 13-12 has been identified as a product released from the enzyme by acid denaturation during steady-state turnover.273 274 Isotopic exchange with 3H in the solvent275 and measurement of 13C isotope effects277 have provided additional verification of the mechanism. The catalytic activity of the enzyme is determined by ionizable groups with pKa values of 7.1 and 8.3 in the ES complex.278... 

At least three ionizable groups on the enzyme are necessary for full catalytic activity. 

Of the three ionizable groups on the free enzyme, two appear to interact directly with the nucleotides (pfiC values 5.4 and 6.5 at 25°, 0.2 M KC1) while one is affected by the association but is not in contact... 

Some data are shown in Fig. 26. The analysis has been carried out on the assumption of two ionizable groups in the enzyme. 

The pH dependence of ka/Ks for a number of substrates is shown in Fig. 26b. The lines through the points have been calculated from Eq. (3) with a single pair of values of Ka and Kb. The fit is quite good in all cases and indicates that the same ionizable groups on the free enzyme are involved in each case, and, in particular, that step 1 (UpU, UpA, and CpA) and step 2 (U > p and C > p) appear to involve the same groups. 

Enzyme activity (kc.M/Km) as a function of pH for three different enzymes. The optimum pH usually is a characteristic of the enzyme and not the particular substrate. Often the pH sensitivity is an indication of an ionizable group at the active site. 

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