PH dependence, of enzymes

The pH dependency of enzyme-catalyzed reactions also exhibits an optimum. The pH optima for enzyme-catalyzed reactions cover a wide range of pH values. Eor instance, the subtihsins have a broad pH optima in the alkaline range. Other enzymes have a narrow pH optimum. The nature of the pH profile often gives clues to the elucidation of the reaction mechanism of the enzyme-catalyzed reaction. The temperature at which an experiment is performed may affect the pH profile and vice versa. 

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

In characterizing the pH dependence of enzyme activity, one often observes (a) a bell-shaped curve in plots of activity versus pH (Fig. 1), or (b) S -shaped activity versus pH curves (either falling from optimal activity or rising to optimal activity) with an inflection point at some... 

Approximations (1) and (2) have been made in the earliest models of development of enzymatic sensors in order to simplify the mathematics. They are both bad the concentration profiles are nonlinear (Caras et al., 1985a Eddowes, 1985) and the pH dependence of enzyme kinetics is an established fact. 

The following questions are important for analysing the pH dependence of enzyme catalysed reactions ... 

Hase (H16) studied the effect of pH on the hydrolysis of acetylcholine by horse serum cholinesterase, and his results have been reanalyzed by Laidler (L5) and extensively discussed by Dixon and Webb (D21). The relationship between pH and the rate of hydrolysis of acetylcholine has been used to obtain information on the structure of the active site of the enzyme (B19, W28). Acetylcholine is a particularly suitable substrate for these studies since it does not change its charge in the pH range studied. Similar pH-activity curves have been obtained using other substrates for cholinesterase (H23, S20, P19). Moreover the pH dependence of enzymic activity varies with the buffer system (K3). By investigating the effect of pH and sodium chloride concentration on the rate of hydrolysis of ben-zoylcholine by human plasma cholinesterase, Kalow (K6) deduced that for this substrate, each enzyme molecule contains at least two binding sites which differ in their dependence on pH. Michaelis constants and maximum hydrolysis velocities were measured for each of the two binding sites, and pK values of the enzyme-substrate complexes were found to be 5.2, 6.7, and 9.2 for one site, and 5.2, 7.0, 8.4, and 8.8 for the other. 

Information in support of this hypothesis has been obtained in detailed studies on the inactivation kinetics and mechanism of pig and beef liver MAO-A by A -cyclopropyl-A-arylalkylamines. The structures and some kinetic properties of this second group of MAO inhibitors are summarized in Table III. The inactivation characteristics of the A/-cyclopropylamines are generally similar to those of the propargylamines (1) time-dependent, first-order loss of enzyme activity, saturation kinetics, and protection from inactivation by substrate or product (2) pH-dependent rate of inactivation corresponding to the pH dependence of enzyme activity (3) little activity recovery after exhaustive dialysis (4) partitioning between normal product formation and inactivation and (5) time-dependent conversion of the covalently bound FAD cofactor from the oxidized to a reduced form, which is fairly resistant to reoxidation. An important differ-... 

Evaluation of stability and catalytic properties of the immobilized system must take into account possible pH differences between the inner core of the fiber, where the reaction takes place, and the bulk of the feed solution. The production of compounds which alter the pH, like ammonia produced from urea via immobilized urease,48 and the partition properties in hollow fiber membranes can result in creating such pH gradients. Experimentally, these differences produce more or less pronounced shifts in the optimum pH dependence of enzyme activity relative to its free form dependence and thereby affect the activity of the enzyme at work.2 3 48... 

The factors involved in the efficient action of enzymes are by no means understood. A considerable amount of work must be done to clarify this problem, and many suggestions have been made. An important clue is provided by the pH dependence of enzyme action, which we have considered earlier in this chapter. We saw that the active form of the enzyme is the intermediate ionic form, which contains a basic group (the —COO " group in the scheme on p. 442) and an acidic group (the —in the scheme). For example, consider the hydrolysis of an ester,... 

We will consider the following reaction scheme (Figure 6.51), which accounts for the pH dependence of enzyme action... 

McLaren, A. D. 1957. Concerning the pH dependence of enzyme reactions on cells, particulates and in solution. Science 125 697. 

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. 

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