Kinetic solvent isotope effect

R. Hille and R.F. Anderson, Coupled electron/proton transfer in complex flavoproteins — solvent kinetic isotope effect studies of electron transfer in xanthine oxidase and trimethylamine dehydrogenase. J. Biol. Chem. 276, 31193-31201 (2001). 

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

Solvent Kinetic Isotope Effects in Enzyme Reactions... 

An S Ar (nucleophilic substitution at aromatic carbon atom) mechanism has been proposed for these reactions. Both nonenzymatic and enzymatic reactions that proceed via this mechanism typically exhibit inverse solvent kinetic isotope effects. This observation is in agreement with the example above since the thiolate form of glutathione plays the role of the nucleophile role in dehalogenation reactions. Thus values of solvent kinetic isotope effects obtained for the C13S mutant, which catalyzes only the initial steps of these reactions, do not agree with this mechanism. Rather, the observed normal solvent isotope effect supports a mechanism in which step(s) that have either no solvent kinetic isotope effect at all, or an inverse effect, and which occur after the elimination step, are kinetically significant and diminish the observed solvent kinetic isotope effect. 

A solvent kinetic isotope effect (SKIE) of 0.44 from solvolysis in CD3CN-H2O versus CD3CN-D2O mixtures was in line with predicted values for the protonation-dissociation mechanism for which the SKIE should be between 0.48 and 0.33 (Figure 18a... 

In Guo, after the very fast protonation of the electron adduct by water at the heteroatom [k > 107 s 1, von Sonntag 1991 Candeias et al. 1992 at 0(6), N(3) or N(7), cf. reaction (180)], a rapid transformation occurs [reaction (181) k in H20) = 1.2 x 106 s k(in D20) = 1.5 x 10s s 1] which is also catalyzed by phosphate buffer (k = 5.9 x 107 dm3 mol-1 s 1) which has been attributed to a protonation at C(8) (Candeias et al. 1992). This assignment is based upon solid-state EPR data, where C(8)-H--adduct is the thermodynamically most stable H -adducl radical (Rakvin et al. 1987 for DFT calculations see Naumov and von Sonntag, unpubl. results). The high solvent kinetic isotope effect of ku/ko = 8 is a strong indication that a proton is transferred in the rate-determining step. The magnitude of the rate of phosphate buffer catalysis points to a protonation at carbon (for a similar reaction observed with the Thy radical anion see Table 10.20). The C(8)-H -ad-duct has a pKa value of 5.4 [equilibrium (182)]. 

Gold and Grist (1972) concluded that strongly basic anions perturb the water molecules that are hydrogen bonded to them, thereby contributing to inverse solvent kinetic isotope effects, but they did not provide evidence for a molecular explanation. 

Schowen (1972) used the Swain-Bader theory to explain solvent kinetic isotope effects on SN2 reactions involving halides, in terms of the numer of water molecules solvating a halide ion in the transition state (assumed to be three) vs. the number solvating it in the bulk (four). The contribution of a single halide-water hydrogen bond was also taken to depend on the partial charge on the halide, which could be consistent with either theory. 

Wolthers, Kirsten R., Schimerlik, Michael I. (2002) Neuronal nitric oxide synthase substrate and solvent kinetic isotope effects on the steady-state kinetic parameters for the reduction of 2, 6-dichloroindophenol and cytochrome C3+, Biochemistry 41, 196-204. 

The carboethoxy stabilized secondary enamines, 25 and 26, were studied by Guthrie and Jordan68. In the absence of buffer, and at pH 5 to 6, acid catalysis is evident and a solvent kinetic isotope effect, (kH+/kD+) = 2.3, is found for 25. These results clearly support rate-controlling C-protonation of the enamine the catalytic constants are included in Table 9. Both 25 and 26 show general-acid catalysis of hydrolysis in the pH... 

The reactivity of a large number of amides (of widely differing structure) in acid-catalysed nitrosation has been determined (Mirvish, 1975). There appears to be no correlation between the rate constants and the basicity of the amides. In this case (Scheme 7 X = H O) it is not known what the effective rate-limiting step is. Further studies in this area would be helpful, particularly using the solvent kinetic isotope effect. 

Although at pH 8 the electron distribution favours the formation of flavin semiquinone and reduced iron-sulfur center, the magnetic moments of the two redox centers do not interact. At pH 10, however, 2-electron-reduced TMADH exhibits the EPR spectrum diagnostic of the spin-mteracting state. In a more detailed analysis using the pH-jump technique, the interconversion of three states of TMADH [state 1, dihy-droflavin-oxidised 4Fe-4S center (formed at pH 6) state 2, flavin semi-quinone-reduced 4Fe-4S center (formed at pH 8) state 3, spin interacting state (formed at pH 10)] were studied in both H2O and D2O (Rohlfs et al., 1995). The kinetics were found to be consistent with a reaction mechanism that involves sequential protonation/deprotonation and electron transfer events (Figure 6). Normal solvent kinetic isotope effects were observed and proton inventory analysis revealed that at least one proton is involved in the reaction between pH 6 and 8 and at least two protons are involved between pH 8 and 10. At least three protonation/... 

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. 

Kim, J. H., Ryan, M. G., Knaut, H., and Hille, R., 1996, The reductive half-reaction of xanthine oxidase a pH dependence and solvent kinetic isotope effect study, J. Biol. Chem. 271 677196780. 

Solvent isotope effects. Much mechanistic information can be obtained about reactions involving proton transfer from solvent kinetic isotope effects, particularly in solvents of mixed isotopic composition. For practical reasons work is essentially confined to H/D effects, especially those in water. Unlike ordinary primary hydrogen isotope effects, solvent isotope effects have to take into account a host of exchangeable sites, subject to equilibrium as well as kinetic isotope effects. A key concept is that of the fractionation factor, (p, which is the deuterium occupancy of a site in a 1 1 H2O/D2O mixture more formally it is defined by equation l.l ... 

Solvent kinetic isotope effects (SKIEs) in H2O/D2O mixtures on the reaction of /)NPP catalyzed by calcineurin gave a small normal value of 1.35. Proton inventory and fractionation data are consistent with a mechanism involving a single proton transfer from a metal-bound water, although due to the small KSIE value and the inherent experimental error of the proton inventory technique, the participation of a second proton could not he excluded. Further information has been furnished by heavy-atom isotope effects. Reaction of NPP catalyzed by APP shows that phosphoryl transfer is fully rate limiting. However, for calcineurin the... 

The second-order rate constants and solvent kinetic isotope effects for acid-catalyzed hydration are given below for several 2-substituted 1,3-butadienes. The products are a mixture of 1,2- and 1,4-addition. What information do these data provide about the mechanism of the reaction ... 

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