Experimental Evidence for Extensive Tunneling of Hydrogen in the Lipoxygenase Reaction: Implications for Enzyme Catalysis

摘要

Hydrogen tunneling has been shown to contribute to the reactions catalyzed by alcohol dehydrogenases from yeast and horse liver, bovine serum amine oxidase, and monoamine oxidase B~4 under ambient conditions. We now report evidence for extensive tunneling of both H and D in the oxidation of linoleic acid catalyzed by soybean lipoxygenase (SBL). Recent investigations of steady state kinetic isotope effects in the SBL reaction have indicated the largest deuterium isotope effect reported for a biological system (ka/ko = 48 at 25 °C). A kinetic picture has emerged in which the rate-limiting step changes from one which is partially limited by substrate binding (at room temperature) to one which is fully rate limited by the C-H bond abstraction (above 32°C). The temperature dependence of Jtcai for the steady state oxidation of linoleic acid by soybean lipoxygenase at 32 °C and above is presented in Figure 1. The maximum isotope effect is 56 ± 5 at 32 °C, decreasing slightly with further increases in temperature as anticipated for an isolated C—H bond-cleavage step. The data indicate almost parallel lines with enthalpies of activation of 1.6 and 1.2 kcal/mol for D-LA and H-LA, respectively (Table 1). Extrapolation of the data in Figure la to infinite temperature ieads to an isotope effect on the Arrhenius prefactor which is enormous (AH/AD = 27 ± 15). This behavior, which is in marked contrast to classical behavior that predicts AH/AD approx =1, is a consequence of the fact that the isotope effects are large yet almost independent of temperature.