Kinetic isotope effects substrate dependence

Kinetic data exist for all these oxidants and some are given in Table 12. The important features are (i) Ce(IV) perchlorate forms 1 1 complexes with ketones with spectroscopically determined formation constants in good agreement with kinetic values (ii) only Co(III) fails to give an appreciable primary kinetic isotope effect (Ir(IV) has yet to be examined in this respect) (/ ) the acidity dependence for Co(III) oxidation is characteristic of the oxidant and iv) in some cases [Co(III) Ce(IV) perchlorate , Mn(III) sulphate ] the rate of disappearance of ketone considerably exceeds the corresponding rate of enolisation however, with Mn(ril) pyrophosphate and Ir(IV) the rates of the two processes are identical and with Ce(IV) sulphate and V(V) the rate of enolisation of ketone exceeds its rate of oxidation. (The opposite has been stated for Ce(IV) sulphate , but this was based on an erroneous value for k(enolisation) for cyclohexanone The oxidation of acetophenone by Mn(III) acetate in acetic acid is a crucial step in the Mn(II)-catalysed autoxidation of this substrate. The rate of autoxidation equals that of enolisation, determined by isotopic exchange , under these conditions, and evidently Mn(III) attacks the enolic form. 

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. 

Human type II inosine monophosphate dehydrogenase catalyses NAD-dependent conversion of inosine monophosphate (IMP) into xanthosine monophosphate (XMP) measurements of the primary kinetic isotope effect using [ H]IMP suggest that both substrates (IMP and NAD) can dissociate from the enzyme-substrate complex therefore, the kinetic mechanism is not ordered. NMR studies indicate hydride transfer to the B or pro-S face of the nicotinamide ring of NAD, while kinetic studies suggest... 

The most widely accepted mechanism for electrophilic aromatic substitution involves a change from sp2 to sps hybridization of the carbon under attack, with formation of a species (the Wheland or a complex) which is a real intermediate, i.e., a minimum in the energy-reaction coordinate diagram. In most of cases the rate-determining step is the formation of the a intermediate in other cases, depending on the structure of the substrate, the nature of the electrophile, and the reaction conditions, the decomposition of such an intermediate is kinetically significant. In such cases a positive primary kinetic isotope effect and a base catalysis are expected (as Melander43 first pointed out). 

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... 

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. 

The analysis and interpretation of kinetic isotope effects (KIE) from the carbonyl carbon has been used to postulate SET processes for the first step in the reactions between ketones and MeLi or Mc2CuLi however, the ratedetermining steps within the overall mechanistic scheme in Eq. (5) depend on the steric and electronic properties of the substrate [63]. 

Methylamine dehydrogenase (MADH) catalyses the oxidative conversion of primary amines to aldehyde and ammonia. This enzyme is found in several methylotrophic bacteria that use amines as their principal source of carbon and energy. Experiments show unusually large primary kinetic isotope effects for the rate-limiting proton transfer step in the MADH reaction. These results imply that there is a large contribution to the proton transfer reaction from quantum tunnelling. Experiments also show that there is almost no dependence of the primary kinetic isotope effect on temperature for the methylamine substrate.151... 

An important consequence of the for one substrate measured by its inhibition of the hydrolysis of a second being its is that the ratio of rates of two competing substrates depends only on their kcatIKm ratio, at all substrate concentrations. This is shown in eqn. (5.23). It is particularly germane to the measurement of kinetic isotope effects from the changes in isotopic enrichment in substrate or product such measurements always give the effect on... 

The simplest case conceptually is when kcat is fully rate limited by chemistry and, therefore, probes of chemistry (such as isotope effects) are revealed in kcat-This limit is realized in recombinant soybean lipoxygenase-1 (SLO) and its mutants, greatly facilitating the study of C-H cleavage in this enzyme [7-9]. The study of chemistry in this case simply requires that kcat can be faithfully measured as a function of external perturbation, such as temperature, pH, or substrate deut-eration. It is essential to show substrate saturation ([S] Km) under all conditions, however, and this requirement can present experimental limitations. The principal probe for tunneling in enzymes in this kinetic case is the magnitude and temperature dependence of noncompetitive kinetic isotope effects, kcat = kcat(H)/fecat(o)-... 

Large primary kinetic isotope effects have been measured for the H-atom transfer steps from substrate to dAdo and from dAdo to the product radical in a number of AdoCbl-dependent enzymes as indicated in Table 19.1. In methylmalonyl-CoA mutase, the steady-state deuterium isotope effect is 5-6 in the forward direction, and the intrinsic isotope effect of step (i) in Scheme 19.3 is masked by the kineti-cally coupled but slower later steps [37-39]. The steady-state tritium kinetic isotope effect kii/kj) in the forward direction has been reported to be 3.2 [38]. Note that the experiments with deuterium were performed with a fully deuterated methyl group, while those with tritium were carried out at the trace level and correspond to a single isotopic atom therefore these two isotope effects should not be directly compared. For the reverse reaction, the deuterium kinetic isotope effect is also par-... 

The means by which enzymes facilitate the homolytic cleavage of the Co-C5 bond has been addressed in detail in studies of MCM. This process can be kinetically monitored by the spectral change from Co(III) in coenzyme B12 to that of cob(II)alamin. This spectral change depends on the addition of the substrate to the complex of MCM and adenosylcobalamin. The kinetic barrier to bond cleavage is lowered by 17 kcalmol . " " Moreover, the rate of this change displays a kinetic isotope effect of >20 (Fh/F ) when the deuterated substrate is employed. " It was concluded that Co-C5 bond cleavage and hydrogen abstraction from the substrate are kinetically coupled. This effect has been reported for other coenzyme Bj2-dependent reactions as well. 

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