Kinetic isotope effects enzymes effect determination using

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. 

Just as in the preceding examples, early indications of tunneling in enzyme-catalyzed reactions depended on the failure of experiments to conform to the traditional expectations for kinetic isotope effects (Chart 3). Table 1 describes experimental determinations of -secondary isotope effects for redox reactions of the cofactors NADH and NAD. The two hydrogenic positions at C4 of NADH are stereochemically distinct and can be labeled individually by synthetic use of enzyme-catalyzed reactions. In reactions where the deuterium label is not transferred (see below), an... 

It should be noted that there is a kinetic isotope effect on the normal reaction (9.11) when the a-deuterated compound is used as the substrate. A similar effect is found when the deuterated suicide inhibitor is used. Thus, both reactions involve a proton transfer in the rate-determining step of the reaction. It has also been shown that a sample of the allenic intermediate that is prepared chemically does in fact irreversibly inhibit the enzyme.18... 

The contribution of Paneth and coworkers demonstrates how substrate-enzyme interactions could be explored using experimentally determined kinetic isotope effects and QM/MM calculations. 

In the case of enzymes working via a ternary complex mechanism, we have two extreme cases. The easiest to comprehend is the rapid equilibrium random mechanism (Scheme 5.4) this is the mechanism where the chemistry is most likely to be rate determining and kinetic isotope effects or structure-reactivity correlations are likely to be mechanistically informative. Enzymes acting on their physiological substrates at optimal pH are likely to show a degree of preference for one or the other substrate binding first, but they can often be induced to revert to a rapid equilibrium random mechanism by the use of non-optimal substrates or pH. 

The ability to accurately compute kinetic isotope effects (KIEs) for chemical reactions in solution and in enzymes is important because the measured KIEs provide the most direct probe to the nature of the transition state and the computational results can help rationalize experimental findings. This is illustrated by the work of Schramm and co-workers, who have used the experimental KIEs to develop transition state models for the enzymatic process catalyzed by purine nucleoside phosphorylase (PNP), which in turn were used to design picomolar inhibitors. In principle, Schramm s approach can be applied to other enzymes however, in order to establish a useful transition state model for enzymatic reactions, it is often necessary to use sophisticated computational methods to model the structure of the transition state and to match the computed KIEs with experiments. The challenge to theory is the difficulty in accurately determining the small difference in free energy of activation due to isotope replacements, especially for secondary and heavy isotope effects. Furthermore, unlike studies of reactions in the gas phase, one has to consider... 

Isotope effects have been used to determine whether the hydride transfer from the enzyme cofactor nicotinamide-adenine dinucleotide (NADH) (reaction (43)) takes place as a hydride ion transfer in a single kinetic step or in a multistep reaction via an uncoupled electron and hydrogen transfer. 

The practical usefulness of Equations 11.46 through 11.53 has been demonstrated for the malic enzyme catalyzed conversion of L-malate to pyruvate (Equation 11.72). Table 11.1 lists experimentally determined isotope effects for this reaction. Comparison of carbon kinetic isotope effects for protio and deutero-malate substituted at position 2 (the carbon that undergoes sp3 to sp2 transition) rules out the possibility that the hydride transfer and the decarboxylation events are concerted. This conclusion follows from Equation 11.48 which, for a concerted reaction, predicts that 13(V/K) should be smaller than 13(V/K)D, which is opposite to the order observed experimentally. 

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