Isotope effect, lactate dehydrogenase

Andres, J., Moliner, V. and Safont, V. S. Theoretical kinetic isotope effects for the hydride-transfer step in Lactate Dehydrogenase, J. Chem. Soc. Faraday Trans., 90 (1994),... 

Isotope effects have also been applied extensively to studies of NAD+/NADP+-linked dehydrogenases. We typically treat these enzymes as systems whose catalytic rates are limited by product release. Nonetheless, Palm clearly demonstrated a primary tritium kinetic isotope effect on lactate dehydrogenase catalysis, a finding that indicated that the hydride transfer step is rate-contributing. Plapp s laboratory later demonstrated that liver alcohol dehydrogenase has an intrinsic /ch//cd isotope effect of 5.2 with ethanol and an intrinsic /ch//cd isotope effect of 3-6-4.3 with benzyl alcohol. Moreover, Klin-man reported the following intrinsic isotope effects in the reduction of p-substituted benzaldehydes by yeast alcohol dehydrogenase kn/ko for p-Br-benzaldehyde = 3.5 kulki) for p-Cl-benzaldehyde = 3.3 kulk for p-H-benzaldehyde = 3.0 kulk for p-CHs-benzaldehyde = 5.4 and kn/ko for p-CHsO-benzaldehyde = 3.4. 

A related approach is to study complexes formed with normal NAD+ but with an unreactive second substrate. An example is oxamate, which binds well to lactate dehydrogenase to form stable ternary complexes for which equilibrium isotope effects have been studied.39... 

One of the first measures of how strongly hydrogen bonds can be reflected in isotope effects was the experimental determination of the oxygen isotope effect on binding of an inhibitor, oxamate, to lactate dehydrogenase.7 The inverse isotope effect... 

Gawlita E, Anderson VE (1994) Paneth P Semiempirical calculations of the oxygen equilibrium isotope effect on binding of oxamate to lactate dehydrogenase. Eur. Biophys. J. 23 353-360... 

Gawlita E, Anderson VE, Paneth P (1995) Equilibrium isotope effect on ternary complex formation of [l-lsO]oxamate with NADH and lactate dehydrogenase. Biochemistry 34 6050-6058... 

In the reduction of pyruvate to lactate, isoenzymes of lactate dehydrogenase from pig heart and pig muscle exerted no kinetic deuterium isotope effect (Holbrook and Stinson, 1973). The results reveal that the process involving the movement of the hydrogen nucleus does not constitute the rate-determining step. Here, the isomerization of the substrate-NADH-enzyme ternary complex to an "active complex" is suggested to be the rate-determining step. The question of whether the "active complex" corresponds to a "charge transfer complex" or to a "conformationally distorted complex" remains unsolved. 

Ferrer S, Tunon 1, Marti S, Moliner V, Garcia-Viloca M, Gonzalez-Lafont A, Lluch JM (2006) A theoretical analysis of rate constants and kinetic isotope effects corresponding to different reactant valleys in lactate dehydrogenase. J Am Chem Soc 128(51) 16851-16863... 

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