Kinetic isotope effects intrinsic

NORTHROP ISOTOPE EFFECT METHOD INTRINSIC KINETIC ISOTOPE EFFECT KINETIC ISOTOPE EFFECT Intrinsic viscosity,... 

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. 

Using the various simplifications above, we have arrived at a model for reaction 11.9 in which only one step, the chemical conversion occurring at the active site of the enzyme characterized by the rate constant k3, exhibits the kinetic isotope effect Hk3. From Equations 11.29 and 11.30, however, it is apparent that the observed isotope effects, HV and H(V/K), are not directly equal to this kinetic isotope effect, Hk3, which is called the intrinsic kinetic isotope effect. The complexity of the reaction may cause part or all of Hk3 to be masked by an amount depending on the ratios k3/ks and k3/k2. The first ratio, k3/k3, compares the intrinsic rate to the rate of product dissociation, and is called the ratio of catalysis, r(=k3/ks). The second, k3/k2, compares the intrinsic rate to the rate of the substrate dissociation and is called forward commitment to catalysis, Cf(=k3/k2), or in short, commitment. The term partitioning factor is sometimes used in the literature for this ratio of rate constants. 

If the overall reaction rate is controlled by step three (k3) (i.e. if that is the rate limiting step), then the observed isotope effect is close to the intrinsic value. On the other hand, if the rate of chemical conversion (step three) is about the same or faster than processes described by ks and k2, partitioning factors will be large, and the observed isotope effects will be smaller or much smaller than the intrinsic isotope effect. The usual goal of isotope studies on enzymatic reactions is to unravel the kinetic scheme and deduce the intrinsic kinetic isotope effect in order to elucidate the nature of the transition state corresponding to the chemical conversion at the active site of an enzyme. Methods of achieving this goal will be discussed later in this chapter. 

The principal goal of most studies of kinetic isotope effects on enzymatic reactions is to deduce intrinsic rate constants, which, in turn, can be correlated with the geometric features, that is the structure, of the corresponding transition states. Formal kinetics provides several options for reaching this goal. For example, as we have seen above, changes in concentration can diminish the commitment to the point where the KIE experimental value corresponds directly to the intrinsic kinetic... 

If the isotope sensitive step is reversible the equations get more complicated and cannot be solved explicitly for the intrinsic isotope effects (unless Cf = 0, or the equilibrium isotope effect is unity). The last two equations in Equation 11.48 demonstrate that a normal deuterium kinetic isotope effect diminishes the apparent commitment if both isotopes are present. Thus 13(V/K) is smaller than 13(V/K)d when both isotope effects are related to the same step. 

Isotope effects on both the carbon and hydrogen of the breaking C-H bond have been measured. However, for this reaction both forward and reverse commitments are sizable so the three equations corresponding to Equation 11.48 have four unknowns the forward and reverse commitments and two intrinsic isotope effects. Measurements of the secondary deuterium kinetic isotope effect (at position 4 of nicotinamide ring of NADP+) and the carbon kinetic isotope effect with the secondary position deuterated introduce two additional equations, but only one more unknown ... 

Equation 11.74 allows for an explicit solution for all five unknowns. The intrinsic values obtained are listed in Table 11.2 together with the experimental ones. In addition to these intrinsic values of kinetic isotope effects to be used in further analysis of the transition state structure, the commitments were calculated as Cf = 0.8 0.3 and cr = 0.5 0.3. 

Table 11.6 Observed and intrinsic kinetic isotope effects on the glucose-6-phosphate dehydrogenase reaction in D2O (Cleland, W. W. in Cook, P. F., Ed., Enzyme Mechanism from Isotope Effects CRC Press, Boca Raton FL, 1991. Hermes, J. D. and Cleland, W. W. J. Am. Chem. Soc. 106, 7263 (1999))...

The haloalkane dehalogenase DhlA mechanism takes place in two consecutive Sn2 steps. In the first, the carboxylate moiety of the aspartate Aspl24, acting as a nucleophile on the carbon atom of DCE, displaces chloride anion which leads to formation of the enzyme-substrate intermediate (Equation 11.86). That intermediate is hydrolyzed by water in the subsequent step. The experimentally determined chlorine kinetic isotope effect for 1-chlorobutane, the slow substrate, is k(35Cl)/k(37Cl) = 1.0066 0.0004 and should correspond to the intrinsic isotope effect for the dehalogenation step. While the reported experimental value for DCE hydrolysis is smaller, it becomes practically the same when corrected for the intramolecular chlorine kinetic isotope effect (a consequence of the two identical chlorine labels in DCE). 

Such considerations raise the concept of the intrinsic kinetic isotope effect—the effect of isotopic substitution on a specific step in an enzyme-catalyzed reaction. The magnitude of an intrinsic isotope effect may not equal the magnitude of an isotope effect on collective rate parameters such as Vmax or Emax/ m, unless the isotopi-cally sensitive step is the rate-limiting or rate-contributing step. To tackle this problem, Northrop extended the kinetic theory for primary isotope effects in enzyme-catalyzed reactions. His approach can be illustrated with the following example of a one-substrate/two-intermedi-ate enzyme-catalyzed reaction ... 

Isotope effects have also been applied extensively to studies of NAD+/NADP+-linked dehydrogenases. We typically treat these enzymes as systems whose catalytic rates are limited by product release. Nonetheless, Palm clearly demonstrated a primary tritium kinetic isotope effect on lactate dehydrogenase catalysis, a finding that indicated that the hydride transfer step is rate-contributing. Plapp s laboratory later demonstrated that liver alcohol dehydrogenase has an intrinsic /ch//cd isotope effect of 5.2 with ethanol and an intrinsic /ch//cd isotope effect of 3-6-4.3 with benzyl alcohol. Moreover, Klin-man reported the following intrinsic isotope effects in the reduction of p-substituted benzaldehydes by yeast alcohol dehydrogenase kn/ko for p-Br-benzaldehyde = 3.5 kulki) for p-Cl-benzaldehyde = 3.3 kulk for p-H-benzaldehyde = 3.0 kulk for p-CHs-benzaldehyde = 5.4 and kn/ko for p-CHsO-benzaldehyde = 3.4. 

Recently, we have modeled9 intrinsic carbon kinetic isotope effects on the ornithine decarboxylase-catalyzed decarboxylations. Decarboxylations occur from the pyridoxal 5 -phosphate (PLP) - substrate complexes. These reactions provide a good model case since a number of 13C kinetic isotope effects for the wild-type enzyme and its mutants, as well as for physiological and slow substrates, have been reported.10 Using AM1/CHARMM/MD calculations on nearly 18000-atom models... 

Sicinska D, Truhlar DG, Paneth P (2005) Dependence of transition state structure on substrate the intrinsic C-13 kinetic isotope effect is different for physiological and slow substrates of the ornithine decarboxylase reaction because of different hydrogen bonding structures. J. Am. Chem. Soc. 127 5414-5422... 

An increased kinetic isotope effect at a higher temperature is not compatible with a simple reaction but only with a mechanism in which a pre-equilibrium is shifted at higher temperatures to reacting species, whose intrinsic isotope effect is higher. The inconstancy of the kinetic order n and of the thermodynamic parameters as functions of [HA] and T, leads to analogous conclusions. The evaluation of all experimental results is compatible with mechanism (68). Formation of the diazoamino compound (N-coupling) takes place throu two intermediates, a 1 1 addition complex (HAArNj )n and the N-[Pg.50]

Denu, J. M., and Fitzpatrick, P. F., 1994, Intrinsic primary, secondary, and solvent kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase evidence against a concerted mechanism. Biochemistry 33 400194007. 

Similar results have been obtained by Baciocchi for the deprotonation of a-substituted 4-methoxytoluenes by 2,6-lutidine and NOs in acetonitrile [145]. In this study, the same values of the Bronsted coefficient (a = 0.24), and of the deuterium kinetic isotope effect (kn/kD = 2.0 for 4-methoxytoluene radical cation) have been obtained with the two bases these results point again towards a highly asymmetric transition state with a very small amount of C-H bond cleavage. Moreover, values of 0.53 and 0.66 eV have been calculated for the intrinsic barrier of the reactions of the radical cations with NO3" and 2,6-lutidine, respectively, again comparable with those observed for acid-base reactions involving carbon acids [140, 141]. 

Table 5.2 VjK) kinetic isotope effects on enzymic nucleoside bond cleavage (intrinsic-position is given.

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