Intermediates reactions, isotope fractionation

More likely, there are ephemeral intermediate species with short residence times, and the reaction proceeds in several steps with several intermediates. In such a reaction pathway, changes in the relative rates of the reaction steps can result in changes in the fractionation. Furthermore, there may be multiple pathways by which a chemical transformation can occur. For example, transformation of Se(IV) to Se(0) could proceed via simple abiotic reaction, or via uptake of FlSeOj by a plant, reduction to Se(-ll) within the plant, incorporation into amino acids, death and decay of the plant, release of the Se(-II), and oxidation to Se". The overall transformation, from Se(lV) to Se(0), is the same, but because the two reaction pathways differ greatly, the overall isotopic fractionation may be greatly different. 

This may seem paradoxical, as the kinetic isotope effect induced by S-O bond breakage still exists. How can the overall reaction have little isotopic fractionation when one step within it has a large kinetic isotope effect The key to understanding this is in the isotopic composition of the intermediate species in the reaction chain. An intermediate may become enriched in heavier isotopes if the next step in the reaction chain preferentially consumes lighter isotopes. In the hypothetical case described above, at steady state the sulfate within the cell is enriched in the heavy isotope by an amount equal to the kinetic isotope effect occurring at step 2. Thus, the isotopic composition of the flux of S through step 2 is the same as that of the flux of S into the cell and the kinetic isotope effect occurring at step 2 has no effect on the overall isotopic fractionation. 

The biochemical pathway of both assimilatory and dissimilatory sulfate reduction is illustrated in Figure 1. The details of the dissimilatory reduction pathway are useful for understanding the origin of bacterial stable isotopic fractionations. The overall pathways require the transfer of eight electrons, and proceed through a number of intermediate steps. The reduction of sulfate requires activation by ATP (adenosine triphosphate) to form adenosine phosphosulfate (APS). The enzyme ATP sulfurylase catalyzes this reaction. In dissimilatory reduction, the sulfate moiety of APS is reduced to sulfite (SO3 ) by the enzyme APS reductase, whereas in assimilatory reduction APS is further phosphorylated to phospho-adenosine phosphosulfate (PAPS) before reduction to the oxidation state of sulfite and sulfide. Although the reduction reactions occur in the cell s cytoplasm (i.e., the sulfate enters the cell), the electron transport chain for dissimilatory sulfate reduction occurs in proteins that are peiiplasmic (within the bacterial cell wall). The enzyme hydrogenase... 

In subaerial C3 plants substrate and reactant (s and r, respectively, in Fig. 5.56) for photosynthesis are both gaseous (atmospheric) C02, which flows through the Calvin cycle (the dark reactions of photosynthesis see Box 1.10) to yield simple carbohydrates (p), which are in turn the source of various metabolic intermediates. The source of the intracellular (kinetic) isotopic fractionation during C fixation is the enzyme rubisco (D-ribulose 1,5-diphosphate carboxylase/oxygenase). There is also an isotopic fractionation resulting from the passage of C02 into the cell. Passive diffusion of C02, at a rate , favours 12C, but the fractionation is small... 

Figure 6.12 Free energy correlation (shown schematically) for the H and D zero-point vibrations for a degenerate stepwise double hydrogen transfer reaction according to Eq. (6.31), where secondary kinetic isotope effects and isotopic fractionation between the initial and the intermediate state were neglected. Adapted from Ref [18c],...

In Fig. 6.14(b) the two-barrier or stepwise transfer Arrhenius diagrams are plotted, where it was assumed that the secondary isotope effects of dissociation and neutralization are small, i.e. equal to 1. In addition, absence of isotopic fractionation is assumed, i.e. (j> = 1. In this case, k /k is equal to the kinetic isotope effects P(j = Pj, of the dissociation and neutralization steps. When these isotope effects are large, which is the case at low temperatures, kUD DD jg equal to 2. The statistical factor arises from the fact that in the DD reaction D is transferred in both steps. Therefore, when the intermediate is reached, return to the reactant as well as reaction to the product occurs with equal probability. By contrast, there is no internal return in the HD reaction which exhibits only a single rate-limiting... 

Kuo and Rose showed that the proton that is removed is retained by the enzyme (67). Stubbe and Abeles prepared an alternative substrate in which fluoride elimination competes with carboxylation 68, 69). Neither result defines the mechanism, but they do show that it is likely that the carbanion derived from the substrate is generated as an intermediate and therefore the reaction is not concerted. Definitive results come from double-isotope fraction studies by O Keefe and Knowles (70) and by Cleland and co-woricers (71). As described for Claisen enzymes, this methodology tests whether processes occur in one or two steps. Labeling of the carboxyl to be transferred with carbon-13 and the proton to be transferred as deuterium provided the means to do this test. The results indicate clearly that proton removal from the substrate to generate the carbanion and transfer of the carboxyl occurs in distinct steps. The resulting attack of the carb-... 

Here, and are, respectively, the isotope fractions in initial reactant and in reaction product, and a is the isotope fraction in the intermediate substance that leads directly to the product. 

Vogler EA, Hayes JM (1979) Carbon isotopic fractionation in the Schmidt decarboxylation evidence for two pathways to products. J Org Chem 44 21) 3682—3686 Richard JP, Amyes TL, Lee Y-G, Jagannadham V (1994) Demonstration of the chemical competence of an iminodiazonium ion to serve as the reactive intermediate of a Schmidt reaction. J Am Chem Soc 116(23) 10833-10834... 

Biochemical reaction pathways are often dependent on the mass of an element at a particular position in a molecule. The result mostly is to favour the lighter isotopic species in the formation of the product. Often, exact information about the intermediate activated complex between reactants and products is not available. This makes calculation of rate constants and hence isotope fractionations from first principles difficult. 

Further evidence for the Aa11 mechanism was obtained from a solvent kinetic isotope study. The theoretical kinetic isotope effects for intermediates in the three reaction pathways as derived from fractionation factors are indicated in parentheses in Scheme 6.143,144 For the Aa11 mechanism (pathway (iii)) a solvent KIE (/ch2o A d2o) between 0.48 and 0.33 is predicted while both bimolecular processes (pathways (i) and (ii)) would have greater values of between 0.48 and 0.69. Acid-catalysed hydrolysis of ethylene oxide derivatives and acetals, which follow an A1 mechanism, display KIEs in the region of 0.5 or less while normal acid-catalysed ester hydrolyses (AAc2 mechanism) have values between 0.6 and 0.7.145,146... 

A significant fraction of H2S produced by dissimilatory processes will also be oxidized by other reactions (Jprgensen, 1982). These intermediate sulfur species (e.g., elemental sulfur) from H2S oxidation may be enriched in 34S, also contributing to the overall 34S signal of H2S (Fry et al., 1988 Canfield and Thamdrup, 1994). Stable sulfur isotopes in... 

---