Bell-shaped pH-dependence

Fumarate hydratase (fumarase), which is discussed in Chapter 13, catalyzes the reversible hydration of fumaric acid to malic acid (Eq. 13-11). It was one of the first enzymes whose pH dependence was studied intensively. A bell-shaped pH dependence... 

The following reaction involves both a general-acid and a general-base catalyst in water. Yet, a bell-shaped loglt bs vs. pH profile is not found. Derive the rate expression and explain why the reaction does not lead to a bell-shaped pH dependence. How is this reaction fundamentally different from the reactions that would show a bell-shaped log it,, vs. pH profile What kind of catalysis would the kinetic expression you derived lead a researcher to propose for this reaction ... 

Metal ion association constants Kassoc (Kassoc = in the various Fe(II)-M(II) derivatives of GpdQ (M = Co, Mn, Zn, Cd) were determined at various pH values. A bell-shaped pH dependence was observed for the Fe(II)Zn(II), Fe(II)-Co(II) and Fe(II)Mn(II) derivatives, which indicates that (at least) two ionisable residues (characterized by pKai and pKa2) in the enzyme contribute to binding of metal ions. An overview of the pKa values of amino acid residues near the active site in GpdQ is shown in Table 3.6. 

The first indication of the importance of proton-transfer reactions in enzyme catalysis come from the observation that the rate of most enzyme-catalysed reactions displays a relatively simply, sigmoidal or bell-shaped pH dependence. Thus enzymatic reactions require a small number of acids in a definite state of ionization. Later mechanistic studies indeed confirmed that in many cases these acids and bases - usually identifiable from the pK values of the pH-rate profile -act as proton donors and proton acceptors in the rate-limiting step of the catalytic process. Since in biological systems enzymatic reactions occur almost invariably near neutrality, where oxonium and hydroxide ion concentrations are at a minimum, it is not surprizing to find that enzymes make extensive use of general acid and general base catalysis. 

Additional features of the chymotrypsin mechanism have been elucidated by analyzing the dependence of the reaction on pH. The rate of chymotrypsin-catalyzed cleavage generally exhibits a bell-shaped pH-rate profile (Fig. 6-20). The rates plotted in Figure 6-20a are obtained at low (subsaturating) substrate concentrations and therefore represent The plot can be... 

Another type of degradation leading to a bell-shaped pH-rateprofile is a successive reaction in which a pH-dependent change in the rate-determining step occurs. Hydrolysis of benzothiadiazines such as hydrochlorothiazide shows a bell-shaped pH-rate profile, as seen in Fig. 75.253 An inflection point observed at pH 8-10 is attributed to ionization of the sulfamide group, whereas one occurring around pH 4.5 results from a change in the... 

The kinetics of PAPs exhibit a bell-shaped pH—rate dependency, typical of acid—base catalysis. NMR data obtained with recombinant human PAP between pH 5.5 and pH 7.1 indicates that pA 2 does not involve a metal ligand, and may instead be due to the ionization of one of the two conserved histidine residues near the active site. It has been proposed that one of these histidine residue acts as a general acid in protonation of the leaving group. ° The other histidine residue (H92 in the human PAP and H202 in the kidney bean PAP ) has been suggested to assist in substrate positioning. 

It has been shown that the rate of formation of oximes is at a maximum at a pH that depends on the substrate but is usually 4, and that the rate decreases as the pH is either raised or lowered from this point. We have previously seen (p. 425) that bell-shaped curves like this are often caused by changes in the rate-determining step. In this case, at low pH values step 2 is rapid (because it is acid catalyzed), and step 1... 

The effect of non-participating ligands on the copper catalyzed autoxidation of cysteine was studied in the presence of glycylglycine-phosphate and catecholamines, (2-R-)H2C, (epinephrine, R = CH(OH)-CH2-NHCH3 norepinephrine, R = CH(OH)-CH2-NH2 dopamine, R = CH2-CH2-NH2 dopa, R = CH2-CH(COOH)-NH2) by Hanaki and co-workers (68,69). Typically, these reactions followed Michaelis-Menten kinetics and the autoxidation rate displayed a bell-shaped curve as a function of pH. The catecholamines had no kinetic effects under anaerobic conditions, but catalyzed the autoxidation of cysteine in the following order of efficiency epinephrine = norepinephrine > dopamine > dopa. The concentration and pH dependencies of the reaction rate were interpreted by assuming that the redox active species is the [L Cun(RS-)] ternary complex which is formed in a very fast reaction between CunL and cysteine. Thus, the autoxidation occurs at maximum rate when the conditions are optimal for the formation of this species. At relatively low pH, the ternary complex does not form in sufficient concentration. 

Particularly interesting examples are also the lipophilicity profiles of ampholytes. Depending on the ratio between the neutral tautomer and the zwitterionic tautomer, the log Dow versus pH profile may be bell-shaped or U-shaped [133] (Figure 4). For zwitterions, the shape of the lipophilicity profile depends upon the structure and conformation of the molecule. If the charged groups are situated in proximity and can interact with each other, the zwitterion might be more hydrophobic than the anionic and the cationic species, resulting in a bell-shaped lipophilicity profile. If, however, intramolecular interactions are not possible for steric reasons, the lipophilicity profile is U-shaped [133],... 

Many rate constants in aqueous solutions are pH or pD sensitive. In particular, enzyme catalyzed reactions often show maxima in plots of pH(pD) vs. rate. The example in Fig. 11.5 is constructed for a reaction with a true isotope effect, kH/kD = 2, and with maxima in the pH(pD)/rate dependences as shown by the bell shaped curves. These behaviors are typical for enzyme catalyzed reactions. When the isotope effect is obtained (incorrectly) by comparing rates at equal pH and pD, the values plotted along the steep dashed curve result. If, however, the rate constants at corresponding pH and pD (pD = pH + 0.5) are employed, a constant and correct value is obtained, kH/kD = 2. Thus for accurate measurements of the isotope effects one must control pH and pD at appropriate values (pD = pH + 0.5 in our example) using a series of buffers. In proton inventory experiments (see below) buffers should be employed to insure equivalent acidities across the entire range of solvent isotope concentration (0 < xD < 1), xD is the atom fraction of deuterium [D]/([H] + [D]). 

Fig. 13. The effect of the minimum ionic strength, / , on the pH-rate profile for a typical enzymatic reaction. Two types of curves are generated Type I, bell shaped type II, monotonically decreasing, depending on the pH of the experiment. Graphing the pH behavior as a function of ionic strength (a and b show the transformation) and applying the / cut-off (c), it can be seen that, if experimental pH at is lower than the pH optimum, a type I curve is obtained. If the experimental pH is greater, a type II is obtained.

The effect of enzymes is strongly dependent on the pH value (see p.30). When the activity is plotted against pH, a bell-shaped curve is usually obtained (1). With animal enzymes, the pH optimum—i.e., the pH value at which enzyme activity is at its maximum—is often close to the pH value of the cells (i.e., pH 7). 

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... 

Plots of the activity of various enzymes as a function of pH give different curves, in some cases bell-shaped. The pH dependence of the activity could be the result of three fundamental effects of H ion concentration ... 

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