Secondary isotope effects transfer

Isotope effect between the HH, HD, DH, and DD isotopomers was used as an important tool to determine the mechanism of the double-proton transfer. For concerted degenerate double-proton transfers in the absence of tunneling, the rule of the geometrical mean (RGM) should hold in good approximation, which states that /chh/ hd = /cdh/ dd-Tunneling may lead to a breakdown of this rule but the relation /chh > hd = dh > dd should remain valid. In the absence of secondary isotope effects the relation /chh HD = DH = 2 /cdd sliould liold for a stepwise pathway, even if tunneling is involved. 

The consequences on the magnitude of the secondary a-deuterium KIE of coupling the motion of the nontransferring a-hydrogen into the reaction coordinate motion, as suggested by Kurz and Frieden, was investigated in some model calculations by Huskey and Schowen (1983). They used two different models to calculate the secondary isotope effects for the hydride transfer reaction (45). 

Just as in the preceding examples, early indications of tunneling in enzyme-catalyzed reactions depended on the failure of experiments to conform to the traditional expectations for kinetic isotope effects (Chart 3). Table 1 describes experimental determinations of -secondary isotope effects for redox reactions of the cofactors NADH and NAD. The two hydrogenic positions at C4 of NADH are stereochemically distinct and can be labeled individually by synthetic use of enzyme-catalyzed reactions. In reactions where the deuterium label is not transferred (see below), an... 

If secondary isotope effects arise strictly from changes in force constants at the position of substitution, with none of the vibrations of the isotopic atom being coupled into the reaction coordinate, then a secondary isotope effect will vary from 1.00 when the transition state exactly resembles the reactant state (thus no change in force constants when reactant state is converted to transition state) to the value of the equilibrium isotope effect when the transition state exactly resembles the product state (so that conversion of reactant state to transition state produces the same change in force constants as conversion of reactant state to product state). For example in the hydride-transfer reaction shown under point 1 above, the equilibrium secondary isotope effect on conversion of NADH to NAD is 1.13. The kinetic secondary isotope effect is expected to vary from 1.00 (reactant-like transition state), through (1.13)° when the stmctural changes from reactant state to transition state are 50% advanced toward the product state, to 1.13 (product-like transition state). That this naive expectation... 

In the following year, Cleland and his coworkers reported further and more emphatic examples of the phenomenon of exaltation of the a-secondary isotope effects in enzymic hydride-transfer reactions. The cases shown in Table 1 for their studies of yeast alcohol dehydrogenase and horse-liver alcohol dehydrogenase would have been expected on traditional grounds to show kinetic isotope effects between 1.00 and 1.13 but in fact values of 1.38 and 1.50 were found. Even more impressively, the oxidation of formate by NAD was expected to exhibit an isotope effect between 1.00 and 1/1.13 = 0.89 - an inverse isotope effect because NAD" was being converted to NADH. The observed value was 1.22, normal rather than inverse. Again the model of coupled motion, with a citation to Kurz and Frieden, was invoked to interpret the findings. 

The overall conclusion drawn by Huskey and Schowen was that a combination of coupled motion and tunneling through a relatively sharp barrier was required to explain the exaltation of secondary isotope effects. They also noted that this combination predicts that a reduction of exaltation in the secondary effect will occur if the transferring hydrogen is changed from protium to deuterium for point A in Fig. 4, the secondary effect is reduced by a factor of 1.09. Experimentally, reduction factors of 1.03 to 1.14 had been reported. For points B, C, and D on the diagram, all of which lack a combination of coupled motion and tunneling, no such reductions in the secondary isotope effect were calculated. 

Kurz, L.C. and Erieden, C. (1980). Anomalous equilibrium and kinetic alpha-deuterium secondary isotope effects accompanying hydride transfer from reduced nicotinamide adenine dinucleotide. J. Am. Chem. Soc. 102, 4198-4203... 

Rickert, K.W. and Klinman, J.P. (1999). Nature of hydrogen transfer in soybean lipoxygenase 1 separation of primary and secondary isotope effects. Biochemistry 38, 12218-12228... 

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

The rate of polymerization in emulsion polymerization is proportional to kg-, where kg is the fhain transfer step on the vinyl group (10). Substituting trideuterovinyl acetate for vinyl acetate raised the rate by a factor of 1.76. When the calculation for the isotope effect on rate is done accurately, taking into account the 3% H on the trideuterovinyl, we find that if the effect is purely on k3, the rate should rise by a factor of 1.69 as compared to 1.76 . 02. This is almost within the experimental error. There may be a very slight secondary isotope effect (23,24) on the propagation and re-ini tation rate constants k2 and k, but it cannot be decided from these data. 

The numerical factors in equations (70) to (73) are again statistical. The factors involving l raised to a power imply kinetic secondary isotope effects. The replacement of one protium atom by one deuterium atom increases the rate constant by i . This inverse isotope effect is qualitatively intelligible since the non-transferred nuclei are more tightly bound in the transition state than they were in the reactants. The existence of the secondary isotope effect also means that the ratio of rate constants in H20 and D20 ( H/fcD) Is not a measure of the primary isotope effect, whereas h o/3 h,do is- To obtain the primary isotope effect it is necessary to divide H/feD by Z2 - (For experimental evaluations of primary and secondary isotope effects see Kreevoy et al.t 1964 ... 

The secondary isotope effects, (fcH/fcD)n, can be defined as all the kinetic isotope effects other than that associated with the transferring proton and is mathematically given by equation (16). They are also... 

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