Intrinsic isotope effect determination

The deuterium isotope effects on chemical shift consists of intrinsic isotope effect (direct perturbation of the shielding of X atom) and equilibrium isotope effect (perturbation of the equilibrium caused by the isotopic substitution). The values of deuterium isotope effects are to some extent independent of chemical shifts and allow determination of the mole fraction of the forms in equilibrium. 

It is not always possible to determine intrinsic isotope effects. However, other useful information about the reaction can still be obtained. Above we assumed a single rate determining step sensitive to each isotope substitution. More frequently, however, the isotope sensitivity is found in different steps. Studies with multiple isotope effects can be used to determine the sequence of steps. To illustrate, a more complicated reaction scheme is needed ... 

As mentioned in Section 11.3.5 for the case where the rate determining step is sensitive to both isotopic species, elucidation of the intrinsic isotope effects is not possible using the equations given thus far (if neither the reverse nor the forward commitment is zero). Even then, however, it is possible to solve for the intrinsic iso-... 

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). 

Fig. 5A The dependence on pH of the deuterium isotope effect in the hammerhead ri-bozyme-catalyzed reaction. Black circles show rate constants in H2O gray circles show rate constants in D2O. Solid curves are experimentally determined curves. The apparent plateau of cleavage rates above pH 8 is due to disruptive effects on the deprotonation of uridine and guanosine residues. Dotted lines are theoretical lines calculated from pKa values of hydrated Mg ions of 11.4 in H2O and 12.0 in D2O and on the assmnption that there is no intrinsic isotope effect (a=kH2o/kD2o=l is the coefficient of the intrinsic isotope effect). The following equation was used to plot the graph of pL vs log(rate) log kobs=log(kmax)-log(l+10

Selected entries from Methods in Enzymology [vol, page(s)] Add-base catalysis [with site-directed mutants, 249, 110-118 altered pH dependencies, 249, 110] commitment to [in determination of intrinsic isotope effects, 249, 343, 347-349 in interfacial catalysis, 249, 598-599 equilibrium isotope exchange in, 249, 443-479 hydrogen tunneling in, 249, 373-397] interfacial [competitive inhibitors, kinetic characterization, 249, 604-605 equilibrium parameters, 249, 587-594 forward commitment to, 249, 598-599 interpretation, 249, 578-586 (constraining variables for high processivity, 249, 582-586 kinetic variables at interface,... 

A method used to determine primary intrinsic isotope effects. In this procedure, three differently labeled substrates are used to react with a labeled cosubstrate and the distribution of the labels in the products is measured. 

In order to determine intrinsic isotope effects of benzylic hydroxylations, the metabolism of different deuterated toluenes was investigated in detail with rat liver microsomes and compared with the chemical radical chlorination of... 

It is clear from Equation 26 that unless the commitments are small, one does not determine the intrinsic isotope effect on the isotope-sensitive step. Two methods have been used to... 

The other method for determining intrinsic isotope effects involves measuring deuterium and isotope effects on the same step (21). Equation 26 is used for primary and secondary deuterium isotope effects, and for isotope effects with unlabeled and primary or secondary deuterated substrates. In the latter cases, the commitments are reduced by the size of the intrinsic deuterium isotope effects, which gives five equations in the five unknowns P k, °k, k, Cf, and Cr. Although the errors for the intrinsic isotope effects are reasonable, those on Cf and Cr are large, but their sum is well determined (22). For irreversible decarboxylations where Cr is small, one needs only the primary deuterium isotope effect and the one with unlabeled and deuterated substrates to solve for P k, k, and Cf. 

Hermes JD, Roeske CA, O Leary MH, Cleland WW. Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase. Biochemistry 1982 21 5106-5114. 

The fourth stage of analysis is determination of transition state structures. Clearly, this requires all of the information from Steps 1-3, but in favorable cases we can use isotope effects in the same fashion as the physical organic chemist to get information on transition state structure. The use of structure-function relationships is not so practical, since specificity problems usually distort the results and make interpretation difficult. We shall describe the methods now available for determining intrinsic isotope effects on bond-breaking steps so that this approach can be applied. 

The above method requires that all isotope effects be on the same step. When this is not the case, ways can still be found to determine intrinsic isotope effects in favorable cases, and this has been done with malic enzyme by using intermediate partitioning (776). 

Once intrinsic isotope effects are determined, one is in a position to deduce transition state stmcture, just as the physical organic chemist does for nonen-zymic reactions. Unfortunately, in many cases workers have assumed, rather than proved, that commitments are zero and intrinsic isotope effects were being looked at. Transition state structures have been investigated in the formate and liver alcohol dehydrogenase reactions as the redox potential of the nucleotide substrate was changed (103, 118). Primary deuterium and C, secondary deuterium, and for formate dehydrogenase 0 isotope effects were determined. In both cases the transition states appeared to be late with NAD and to become earlier as the redox potential of the nucleotide became more positive. So far the conclusions from such studies have been qualitative in nature, and there is room for much more work on these systems. 

The determination of the intrinsic isotope effect is based on the Swain-Schaad relationship (Eq. (17.2)). For diminished isotope effects, a fixed relationship between deuterium and tritium is maintained,... 

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. 

The subscript on kcat in Equation 11.19 abbreviates catalyzed . Vmax is connected with the rate determining step. For desorption much faster than catalysis, ks >> k3, Vmax = k3 [E]0 which is the result found for the simpler Michaelis-Menten mechanism, Section 11.2.1. If, however, ks is commensurate with k3 the intrinsic catalysis is damped by the weighting function ks/(k3+ks). Note that Vmax/KM sees events through the first irreversible step as illustrated in Fig. 11.3. The same is true for the isotope effects. These points are discussed in considerable detail by Northrop (see reading list). 

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