The pH Dependence of Enzyme Catalysis

Most enz5miatic reactions in vivo and in vitro take place in aqueous solutions. Most enz)nnes are extremely sensitive to changes in pH of their environment. Whole-cell extracts, and cmde enzyme preparations in general, are well buffered by enzyme and other polyelectrolyte impurities, but this natural buffering is lost when an enzyme is purified, and must be replaced by artificial buffers. 

It is obvious that, in polymers like proteins, the acidic and basic properties are the consequence of protonation and dissociation of acidic and basic groups in side chains of polypeptides. 

Amino acids Types of group Bronsted character at pH 7.0 piCa  

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Thomas, P.G., Russel, A.J., Fersht, A. Tailoring the pH dependence of enzyme catalysis using protein engineering. Nature 318 375-376, 1985. 

The first practical model for the pH dependence of enzyme catalysis was proposed by Michaehs (Michaehs Davidsohn, 1911). The pH behavior of many enzymes can be interpreted as a first approximation in terms of this model, in which only two ionizable groups are considered. 

One of the most distinctive features of enzymic reactions is their ready occurrence at or near neutrality. This behaviour has given rise to many investigations of the pH-dependence of enzymic reactions. Apart from providing information about the optimum pH of the activity of an enzyme, these studies can provide much information related to the pKa values of ionizing groups involved in catalysis.This specific information might eventually lead to the identification of these ionizable groups. A specific example, that of chymotrypsin is provided below. 

The activity of an enzyme is profoundly affected by pH. Usually, enzymes display a bell-shaped activity versus pH profile (Fig. 6.1). The decrease in activity on either side of the pH optimum can be due to two general causes. First, pH may affect the stabihty of the enzyme, causing it to become irreversibly inactivated. Second, pH may affect the kinetic parameters of the enzymatic reaction It may affect the stability of the ES complex, the velocity of the rate-Mmiting step, or both. The second case is relevant to the discussion in this chapter. Interestingly, the pH dependence of enzyme-catalyzed reactions is similar to that of acid- and base-catalyzed chemical reactions. Thus, it is possible, at least in principle, to determine the pK and state of ionization of the functional groups directly involved in catalysis, and possibly their chemical nature. 

Effect of pH on Lignin Peroxidase Catalysis. The oxidation of organic substrates by lignin peroxidase (Vmax) has a pH optimum equal to or possibly below 2. Detailed studies have been performed on the pH dependency of many of the individual reactions involved in catalysis. The effect of pH on the reaction rates between the isolated ferric enzyme, compounds I or II and their respective substrates has been studied. Rapid kinetic data indicate that compound I formation from ferric enzyme and H2O2 is not pH dependent from pH 2.5-7.5 (75,16). Similar results are obtained with Mn-dependent peroxidase (14). This is in contrast to other peroxidases where the pKa values for the reaction of ferric enzyme with H2O2 are usudly in the range of 3 to 6 (72). 

The currently accepted chemical mechanism for the reaction of RNase A was deduced by an inspired piece of chemical intuition before the crystal structure was solved.194 It was found that the pH-activity curve is bell-shaped, with optimal rates around neutrality. The pH dependence of kQJKM shows that the rate depends upon the ionization of a base of pKa 5.22 and an acid of pATfl 6.78 in the free enzyme, whereas the pH dependence of kcax shows that these are perturbed to pKa values of 6.3 and 8.1 in the enzyme-substrate complex. It was proposed that the reaction is catalyzed by concerted general-acid-general-base catalysis by two histidine residues, later identified as His-12 and His-119 (reactions 16.36 and 16.37). 

The pH dependence of kcatIKM shows that the reaction rate is dependent on an acid of pKa 6 and a base of pKa— 4 in the free enzyme.218 The pATa s of all the carboxyls in hen egg white and turkey lysozymes have been measured by two-dimensional -NMR 219 The values for Glu-35 and As-52 are 6.2 and 3.7, respectively, consistent with the pH dependence of catalysis and their roles as general acids and bases, respectively. 

This sort of problem underlines the need for an unequivocal determination of microscopic ionization constants. As already mentioned, the rate-limiting step in catalysis is controlled by an ionization with a pKa of 7. Cruickshank and Kaplan (30) used a-chymotrypsin as a test enzyme and estimated the pKa of its two histidine residues from the pH dependence of their reaction with trace amounts of tritiated l-fluoro-2,4-dinitro-benzene. From their data, a pKa of 6.8 was assigned to His-57 and 6.7 to His-40, which is not involved in catalysis. This data is consistent with deprotonation of His-57 being the critical ionization for catalysis. 

Static interaction with aspartic acid-158. They have accordingly proposed a mechanism in which histidine-159 participates in the catalytic process as a conjugate acid. The pH-dependence of the acylation and deacylation rate constants on an apparent pK near 4 was considered to be caused by a carboxyl group which in its undissociated state led to an inactive conformation of the enzyme. On the other hand, Allen and Lowe (88) argue that the abnormally low pK of histidine-159 can be attributed to its enclosure by a hemisphere of hydrophobic residues, particularly tryptophan-177. A mechanism which portrays histidine as playing a key role in catalysis is presented in Figure 13, although other mechanisms are not necessarily precluded (87, 89). 

The pH dependence of these systems is complex and not fully understood, however. For instance, the same lipase that when used for hydrolysis showed a typical sigmoidal pH -activity profile exhibited practically no pH dependence when used for catalysis of ester synthesis [66]. Evidently, in the very water poor medium of the esterification, the enzyme does not experience a changed environment. 

Based on the pH dependency of the inactivation rate of rabbit muscle triosephosphate isomerase with glycidol phosphate, the pK for Glu-165 was calculated to be less than 5.5 in one study and 6.0 in another. Since this difference could be due to the complication of a variable affinity of the reagent for the enzyme as a result of the change in ionization state of the reagent over the pH range examined, the pH dependency of inactivation rate was also studied with the strong, monoprotic acid chloroacetol sulfate, another compound which selectively esterifies Glu-165. With this compound, the pK of Glu-165 in the rabbit muscle enzyme was found to be less than 5.0. An exact value could not be determined because of the instability of the enzyme to acid however, with the more stable yeast enzyme, a pKa of 3.9 was calculated. Thus, the acidity of the essential carboxyl group is consistent with its postulated role in catalysis. 

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