Primary isotope effects temperature independence

Thus the primary and secondary isotope effects are all within the semiclassical limits and their relationship is in full accord with the semiclassical Swain-Schaad relationship. There is no indication from the magnitudes of the secondary isotope effects in particular of any coupling between motion at the secondary center and the reaction-coordinate for hydride transfer. Thus the sole evidence taken to indicate tunneling is the rigorous temperature-independence of the primary isotope effects. 

Part of the isotope effect is temperature dependent, but for the primary isotope effect the reaction coordinate motion effect, which is always normal, is temperature independent. The primary isotope effects are almost always normal. Secondary isotope effects can be either normal or inverse. 

These data led to the model already described several times above. The enzyme executes a search for a tunneling sub-state, apparently 13 kcaFmol in energy above the principal state from this state the hydrogen atom tunnels with no further vibrational excitation. Probably motion of the secondary center is coupled into the tunneling coordinate. The result is large, temperature-independent primary and secondary isotope effects in the context of an isotope-independent activation energy. 

Lewis et al. (entry 11 of Table 2) examined the temperature-dependence of isotope effects in the action of both the human enzyme and the soybean enzyme, by measuring the relative amounts of per-protio and per-deuterio-13-hydroperoxy-products by HLPC. The observed effects are, therefore, composed of primary, secondary, and perhaps remote isotope-effect contributions. Isotope effects on fecat/ M for both enzymes (determined by competition between labeled substrates) are increased by high total substrate concentration, an effect previously observed but stiU ill-understood. At 100 /rM substrate, the effects are roughly independent of temperature below about 15 °C, and are about 60 (H/D) for the human enzyme and 100 (H/D) for the soybean enzyme. Above 15 °C, the effects decline to about 50 for the human enzyme and about 60 for the soybean enzyme, perhaps because non-isotope-sensitive steps become more nearly rate-limiting (see Chart 4). 

The primary KIEs on kcat also indicated a transition at 30 °C, below which the primary kn/ko ratio is very temperature dependent, extrapolating to Ah/Ad 1 [24]. This inverse Arrhenius prefactor ratio is predicted within the Bell tunnel correction for a moderate extent of tunneling, and is consistent with an elevated a-secondary RS exponent. Above 30 °C, the primary kn/ko ratio is nearly independent of temperature, resulting in an isotope effect on the prefactor of Ah /Ad = 2 [24]. A tunnel correction would also predict such an elevated Arrhenius prefactors ratio when both H and D react almost exclusively by turmeling however this condition requires a very small activation energy for k at, while a value of = 14 kcal mol is observed [24]. 

It is noteworthy that the Swain-Schaad exponents are temperature independent for both primary and secondary isotope effects at pH 6.1. Two scenarios can be considered. The first is that there is a significant commitment to catalysis which is obscuring the full value of the isotope effect on k st/Km- It is anticipated that, because the primary and secondary exponents are temperature independent, this commitment would be temperature independent. Jonsson et al. have used North-rop s expression [60] for correcting observed isotope effects based on the assumption of a temperature-independent commitment (Eq. (10.22)). A commitment of 0.6 for the oxidation of benzylamine brings the secondary exponent to about 3.3,... 

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