Enzyme reactions isotope kinetic effects

Transition state theory has been useful in providing a rationale for the so-called kinetic isotope effect. The kinetic isotope effect is used by enzy-mologists to probe various aspects of mechanism. Importantly, measured kinetic isotope effects have also been used to monitor if non-classical behaviour is a feature of enzyme-catalysed hydrogen transfer reactions. The kinetic isotope effect arises because of the differential reactivity of, for example, a C-H (protium), a C-D (deuterium) and a C-T (tritium) bond. 

Abstract This chapter introduces the basic principles used in applying isotope effects to studies of the kinetics and mechanisms of enzyme catalyzed reactions. Following the introduction of algebraic equations typically used for kinetic analysis of enzyme reactions and a brief discussion of aqueous solvent isotope effects (because enzyme reactions universally occur in aqueous solutions), practical examples illustrating methods and techniques for studying enzyme isotope effects are presented. Finally, computer modeling of enzyme catalysis is briefly discussed. 

In the remaining part of our presentation of the formal kinetics of enzyme isotope effects a few more complicated examples will be discussed. The methods developed here should be also useful for unraveling other complicated enzyme reactions, and in reading and understanding the modern literature on isotope effects on enzymatic processes. 

Carbon kinetic isotope effects on enzyme-catalyzed decarboxylations are among the most intensively studied enzyme reactions. This is because of the central role that carbon dioxide plays in plant metabolism and also because precise kinetic measurements are relatively easy to obtain since the carbon dioxide liberated in the reaction can be immediately analyzed using isotope ratio mass spectrometry. 

Solvent Kinetic Isotope Effects in Enzyme Reactions (See Also Section 11.4)... 

Solvent Kinetic Isotope Effects in Enzyme Reactions... 

Kinetic Isotope Effects in Enzymic Reactions J. H. Richards... 

Besides obvious participation of protons, hydrids and hydrogen atoms in a chemical reaction in enzymes active sites, two main criteria are used for discrimination of particle involvement in the reaction limiting stage site-directed substitution of chosen enzyme groups and kinetic isotope effects (KIE). 

Although the quantitative aspects of isotope effects are difficult to interpret, qualitative aspects have been of considerable use. Whereas the lack of an isotope effect in an overall enzyme-reaction cannot, in the absence of further kinetic analysis, be used as evidence for any particular mechanism, any isotope effect observed should be explicable by the proposed mechanism. 

Enzyme kinetics is an important tool for assaying enzyme activities and for determining enzyme mechanisms. Although other techniques can provide useful information on enzyme mechanisms, the kinetics has to be the ultimate arbiter because it looks at the reaction while it is taking place. Initial velocity patterns, inhibition patterns, patterns of isotopic exchange, pH profiles, and isotope effects are all kinetic tools that allow one to determine kinetic mechanisms, chemical mechanisms, and transition state structures. 

Northrop DB. Steady-state analysis of kinetic isotope effects in enzymic reactions. Biochemistry 1975 14 2644-2651. 

The variationally optimized transition state geometries were found to be different for transfer of a proton or a deuteron, the first indication of such a difference for an enzyme reaction [67]. Quantum treatment of vibrations was found to be important for the calculation of the rate constant, and variational transition state theory was important for calculating kinetic isotope effects. The... 

The dominant tool to study hydrogen tunneling in an enzyme reaction is the measurement of isotope effects on the chemical step of catalysis via steady-state kinetics experiments. However, steady-state kinetics are often complicated by the contribution of several microscopic steps to the macroscopically observed rates, making it difficult to study the chemical step. The following section introduces basic enzyme kinetics, with a discussion of the macroscopic rate constants kat and kcat/ M and their interpretations. More detailed references on this matter are available [1, 2]. The first concern of the experimentalist is to be able to observe the intrinsic rate of chemistry, thereby allowing probes into the mechanism of hydrogen transfer. 

I 70 Nuclear Tunneling in the Condensed Phase Hydrogen Transfer in Enzyme Reactions Table 10.2. Kinetic isotope effect data for various ADH enzymes ). 

Francisco, W. A., Merkler, D. J., Blackburn, N. J., Klinman, J. P. (1998) Kinetic mechanism and intrinsic isotope effects for the peptidylglycine a-amidating enzyme reaction. Biochemistry 37, 8244-8252. 

In glutamate mutase [43], the forward and reverse steady-state deuterium (kn/ko of 3.9 forward and 6.3 reverse) and tritium kn/kj of 21 forward and 19 reverse) kinetic isotope effects are both suppressed. However large deuterium isotope effects of 28 and 35 in the forward and reverse directions respectively have been observed for cob(ii)alamin formation under pre-steady-state conditions. These large kinetic isotope effects suggest that quantum mechanical tunneling also dominates this enzyme reaction. 

Although D2O chemically resembles H2O in most respects, it is a toxic substance. The reason is that deuterium is heavier than hydrogen thus, its compounds often react more slowly than those of the lighter isotope. Regular drinking of D2O instead of H2O could prove fatal because of the slower rate of transfer of compared with that of H+ in the acid-base reactions involved in enzyme catalysis. This kinetic isotope effect is also manifest in acid ionization constants. For example, the ionization constant of acetic acid... 

A summary of some recently discovered inactivators of E. coli PFL is presented in Table V. Consistent with these compounds acting as mechanism-based or active site-directed inhibitors is the observation of pseudo-first-order inactivation kinetics, substrate protection (by pyruvate or formate for inhibitors that are pyruvate or formate analogs, respectively), and isotope effects on the rates of inactivation by the deuterated analogs. The details of some of these studies, the proposed inactivation mechanisms, and the implications to the normal enzymic reaction are discussed below. 

The limits of sensitivity for detection are also dependent upon the nature of the radioisotope in the substrate as well as the analytical method employed to separate and detect substrate, intermediate, and products. In our experience, radiolabels are particularly effective with a detection limit of 4-5% of the total radiolabeled species comprising intermediate for less energetic isotopes such as or H, and less than 1 % detection limit for more energetic isotopes. If the reaction is reversible and depending on the kinetic pathway, there is also the option of looking for the intermediate in the reverse direction by starting with the product. Enzyme reactions that contain an irreversible step(s) are much more challenging since a fewer number of the options are available. 

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