Enzymatic solvent isotope effect

As a practical matter of cost, studies on solvent isotope effects are usually limited to D/H substituted solvents, although recently a few lsO solvent effects have been measured. Interpretation of enzymatic solvent isotope effects is even more complicated than it is when the isotopic probe is incorporated in the substrate(s). This is because enzyme proteins have many exchangeable protons and, also, this is frequently true for reactants (substrates). Thus the observed isotope effect is the collective result of many different isotopic substitutions, each of which may influence... 

Interpretation of KIEs on enzymatic processes (see Chapter 11) has been frequently based on the assumption that the intrinsic value of the kinetic isotope effect is known. Chemical reactions have long been used as models for catalytic events occurring in enzyme active sites and in some cases this analogy has worked quite well. One example is the decarboxylation of 4-pyridylacetic acid presented in Fig. 10.9. Depending on the solvent, either the zwitterionic or the neutral form dominates in the solution. Since the reaction rates in D20/H20 solvent mixtures are the same (see Section 11.4 for a discussion of aqueous D/H solvent isotope effects), as are the carbon KIEs for the carboxylic carbon, it is safe to assume that this is a single step reaction. The isotope effects on pKa are expected to be close to the value of 1.0014 determined for benzoic acid. This in mind, changes in the isotope effects have been attributed to changes in solvation. 

An S Ar (nucleophilic substitution at aromatic carbon atom) mechanism has been proposed for these reactions. Both nonenzymatic and enzymatic reactions that proceed via this mechanism typically exhibit inverse solvent kinetic isotope effects. This observation is in agreement with the example above since the thiolate form of glutathione plays the role of the nucleophile role in dehalogenation reactions. Thus values of solvent kinetic isotope effects obtained for the C13S mutant, which catalyzes only the initial steps of these reactions, do not agree with this mechanism. Rather, the observed normal solvent isotope effect supports a mechanism in which step(s) that have either no solvent kinetic isotope effect at all, or an inverse effect, and which occur after the elimination step, are kinetically significant and diminish the observed solvent kinetic isotope effect. 

The initially observed perturbation (or equilibrium isotope effect) will disappear as isotopic scrambling (or mixing) subsequently occurs. This is illustrated above for the malic enzyme note the initial displacement of the equilibrium toward malate, followed by readjustment of the system to its flnal equilibrium position. The magnitude of the initial perturbation provides information on the occurrence of kinetic isotope effects and the nature of the rate-limiting step in an enzymatic process. See also Kinetic Isotope Effect Solvent Isotope Effect... 

Solvent isotope effects are a useful diagnostic tool in simple chemical reactions, although they can be variable. For example, it is found that general-base-catalyzed reactions such as equation 2.84 have a kH/kD of about 2, whereas the nucleophilic attack on an ester has a kH/kD of about 1. The isotope effects in enzymatic reactions are more difficult to analyze because the protein has so many protons that may exchange with deuterons from D20.61 Also there may be slight changes in the structure of the protein on the change of solvent. 

The introduction of deuterium in place of proton in water, and its consequent exchange into some positions of enzymes and substrates, produces solvent isotope effect on the kinetic and equilihrium constants associated with the enzymatic reactions. These effects, usually expressed as ratios of the appropriate constants in two isotopic solvents HOH and DOD, are useful in the study of reaction mechanisms (Candour Schowen, 1978 Cook, 1991 Quinn Sutton, 1991). 

The ratio of the rate of a reaction in light water to that in heavy water (or in H2O/D2O mixtures) is referred to as a solvent isotope effect. For example, many enzymatic properties, including catalytic rates, differ when the enzyme is dissolved in D2O rather than in H2O. Solvent isotope effects including those on chemical equilibria have been reviewed, among others, by Arnett and McKelvey (1969), Laughton and Robertson (1969), Schowen (1977), and Conway (1981). 

The light hydrogen isotope protium (H) can be replaced by deuterium (D) in the hydrogenic sites of the water species (i.e. HOD, D2O, H+3O, H+2OD, I>20H, D+sO, etc), under certain circumstances, and as a consequence deuterium may replace protium into some sensitive positions of enzymes and substrates these replacements have been designated as solvent isotope effects (SIE) and usually they affect the kinetic and equilibrium constants associated with the enzymatic reactions. Obviously, these SIE are related to the isotopac solvents and thus... 

The most important piece of evidence for proton transfer in enzymatic systems comes from the application of deuterium oxide kinetic solvent isotope effects. If the enzymatic rate-limiting step involves a proton transfer in the transition state, a solvent isotope effect ( hjoAdzo) of 2 to 3 is observed, in good agreement with values observed in non-enzymatic proton transfer reactions [13]. The... 

Because solvent viscosity experiments indicated that the rate-determining step in the PLCBc reaction was likely to be a chemical one, deuterium isotope effects were measured to probe whether proton transfer might be occurring in this step. Toward this end, the kinetic parameters for the PLCBc catalyzed hydrolysis of the soluble substrate C6PC were determined in D20, and a normal primary deuterium isotope effect of 1.9 on kcat/Km was observed for the reaction [34]. A primary isotope effect of magnitude of 1.9 is commonly seen in enzymatic reactions in which proton transfer is rate-limiting, although effects of up to 4.0 have been recorded [107-110]. 

We hope to apply this method in the near future for enzymatic active centres. Replacement of the polar solvent with a fluctuating macromolecular environment, such as an enzyme, is methodologically a straightforward step but requires a lot of coding efforts. Quantum treatment of the nuclear motion is essential for calculation of the kinetic isotope effects that are of vital importance for enzymology. Very recently the H/D kinetic isotope effect h/ d = 80 was simulated with what is in excellent agreement with the experiment [36]. 

In their 60-year history, kinetic isotope effects (KIEs) have been considered the most useful and sensitive tools for studying the mechanisms and determining the structure of the transition states of chemical and enzymatic reactions.1,2 Since the goal of this chapter is to show how KIEs have been used to learn how various factors such as substituents, the solvent and ion pairing of the nucleophile affect the transition structures of SN2 reactions ... 

Deuterium exchange with the solvent does occur during the reaction, but at a rather slow rate. A symmetrical reaction intermediate must exist, since the rate of incorporation of tritium from the solvent into D-madelate as the substrate yields equimolar amounts of D-and L-product [50]. Thus the data are consistent with the formation of an a-carbanion intermediate with an enzymatic base group acting as the proton acceptor [50]. The proton transfer has to be rate-limiting, as indicated by the approximately 5-fold primary isotope effect for deuterium. In the enzyme-substrate complex, the epimerization occurs with a rate constant of the order of 10 s ... 

In this edition, the content was expanded to cover in more depth several areas, such as organocatalysis enzymatic kinetics nonlinear dynamics solvent effects nanokinetics (structure sensitivity) kinetic isotope effects and polynomial kinetics, to name a few. In addition, a separate chapter on cascade catalysis has been written. 

---