Isotope effects, equilibrium substrate

The lack of a substrate isotope effect suggests very extensive internal return and is readily explained in terms of the fact that conversion of the hydrocarbon to the anion would require very little structural reorganisation. Since koba = k 1k 2/(kLl+k 2) and k 2 is deduced as > k2, then kobs = Kk 2, the product of the equilibrium constant and the rate of diffusion away of a solvent molecule, neither of the steps having an appreciable isotope effect. If the diffusion rates are the same for reactions of each compound then the derived logarithms of partial rate factors (above) become pAT differences between benzene and fluorobenzene hydrogens in methanol. However, since the logarithms of the partial rate factors were similar to those obtained with lithium cyclohexylamide, a Bronsted cor-... 

Secondly, it has been found that the benzidine rearrangement is subject to a solvent isotope effect d2o/ h2o > 1- If a proton is transferred from the solvent to the substrate in a rate-determining step the substitution of protium by deuterium will lead to a retardation in the rate of reaction (primary isotope effect) whereas if a proton is transferred in a fast equilibrium step preceeding the rate-determining step as in... 

The reaction was second order in acid and first order in substrate, so both rearrangements and the disproportionation reaction proceed via the doubly-protonated hydrazobenzene intermediate formed in a rapid pre-equilibrium step. The nitrogen and carbon-13 kinetic isotope effects were measured to learn whether the slow step of each reaction was concerted or stepwise. The nitrogen and carbon-13 kinetic isotope effects were measured using whole-molecule isotope ratio mass spectrometry of the trifluoroacetyl derivatives of the amine products and by isotope ratio mass spectrometry on the nitrogen and carbon dioxide gases produced from the products. The carbon-12/carbon-14 isotope... 

Equation 11.36 recognizes that Hk3/Hk4 corresponds to the equilibrium isotope effect, hK3/4 for the step containing rate constants k3 and k4. The rate ratio k4/k.5 is the commitment for catalysis for the reaction that proceeds from products to substrates, and therefore is called the reverse commitment to catalysis, Cr. Also cf = k3/k2 is the forward commitment to catalysis. Since we have assumed that these steps are the only isotope sensitive ones, HK3/4 corresponds to the overall equilibrium isotope effect, HK. 

A related approach is to study complexes formed with normal NAD+ but with an unreactive second substrate. An example is oxamate, which binds well to lactate dehydrogenase to form stable ternary complexes for which equilibrium isotope effects have been studied.39... 

Rates of acid-catalysed enolization of isobutyrophenone and its ot-d analogue have been measured in H2O and D2O, by bromine scavenging.1403 Results include a solvent isotope effect, ku /kDi, of 0.56, and a substrate isotope effect, h/ d, of 6.2 (both for the enolization reaction). Combination of the data with that for ketonization in D2O140b gives the first isotope effect for the keto-enol equilibrium of a simple ketone e(H20)/ e(D20) = 0.92. The results are discussed in terms of the isotopic fiuctionation factors and the medium effect. 

A study of acid-catalysed enolization and carbon-acid ionization of isobutyrophenone has combined the solvent isotope effect k /kv = 0.56 and substrate isotope effect kH/kD = 6.2 determined for the enolization in H2O and D2O with literature information in order to estimate the solvent isotope effect on the enolization equilibrium, A e(H20)/A e(D20) = 0.92, and on the CH ionization of butyrophenone, kf (R20)/kK(D20) = 5.4.130 This is the first report of an isotope effect on AY forketo-enol equilibrium of a simple aldehyde or ketone. 

It follows from these similarities in solvent properties that equilibrium or rate constants of reactions in which the solvent molecules do not directly participate generally show comparatively small changes when the deuterium content of the medium is altered. This is true even for rates of proton transfer between neutral substrates and acetate ions, which as a rule are reduced by 20-40% on going from H20 to D20 (Bell, 1965). Because of the anionic nature of one of the reactants and of the transition state these reactions are of a type in which solvent-solute interactions through hydrogen bonds are probably particularly large, and yet the solvent isotope effect is fairly small. Reactions in... 

The application of isotope effects studies of reaction mechanism includes comparison of experimental values of isotope effects and predicted isotope effects computed for alternative reaction pathways. On the basis of such analysis some of the pathways may be excluded. Theoretical KIEs are calculated using the method of Bigeleisen and Mayer.1 55 KIEs are a function of transition state and substrate vibrational frequencies. Equilibrium isotope effects are calculated from substrate and product data. Different functionals and data sets are used in these calculations. Implementation of a one-dimensional tunnelling correction into conventional transition-state theory significantly improved the prediction of heavy-atom isotope effects.56 Uncertainty of predicted isotope effect can be assessed from the relationship between KIEs and the distances of formed or broken bonds in the transition states, calculated for different optimized structures.57 Calculations of isotope effects from sets of frequencies for optimized structures of reactants and transition states are facilitated by adequate software QUIVER58 and ISOEFF.59... 

These data have led to the development of a catalytic mechanism, shown in Scheme 6, that has been further refined by kinetic isotope effect (KIE) experiments. Substrate binds to Cu(II), replacing bound solvent. The metal coordination facilitates the deprotonation of the substrate hydroxyl group. The proton is transferred to Tyr495, which dissociates from copper. The temperature and pH dependence of the visible absorption and circular dichroism spectra indicate that galactose oxidase exists as an equilibrium of the Tyr495-Cu(II) form (TyroN) and the protonated Tyr495 state. 

If the proton transfer occurs in a pre-equilibrium step (mechanisms A1 or A2), the kinetic solvent isotope effect, kH/kD, is smaller than 1 in almost all known examples [2, 97] (see Table 6). The value of ftH/fcD is determined by the solvent isotope effect on the protonation equilibrium of the substrate. According to eqns. (22) and (23)... 

The third method for measuring isotope effects is equilibrium perturbation (19). In this method, one adds enzyme to a reaction mixture calculated to be at equilibrium containing a labeled substrate and an unlabeled product. For a normal isotope effect, the unlabeled product reacts faster than the labeled substrate and causes a perturbation from equilibrium. As isotopic mixing takes place, however, the reaction comes back to chemical as well as isotopic equilibrium. The size of the perturbation is used to compute the isotope effect. This method is of intermediate precision, but can be used for isotope effects of 1.03 or greater. The isotope effect that is determined is similar to a V/K one. 

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