Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures

The temperature dependence of steady-state kinetics has been studied with h orse liver alcohol dehydrogenase (HLADH) using protonated and deuterated be nzyl alcohol as substrates in methanol/water mixtures between +3 and -50 de greesC. Additionally, the competitive isotope effects, k(H)/k(T) and k(D)/k (T), were measured. The studies indicate increasing kinetic complexity for wild-type HLADH at subzero temperatures. Consistent with earlier findings a t 25 degreesC [Bahnson et al. (1993) Biochemistry 31, 5503], the F93W mutan t shows much less kinetic complexity than the wild-type enzyme between 3 an d -35 degreesC. An analysis of noncompetitive deuterium isotope effects and competitive tritium isotope effects leads to the conclusion that the react ion of F93W involves substantial hydrogen tunneling down to -35 degreesC. T he effect of methanol on kinetic properties for the F93W mutant was analyze d, showing a dependence of competitive KIEs on the NAD(+) concentration. Th is indicates a more random bi-bi kinetic mechanism, in comparison to an ord ered bi-bi kinetic mechanism in water. Although MeOH also affects the magni tude of the reaction rates and, to some extent, the observed KIEs, the rati o of In k(H)/k(T) to In k(D)/k(T) for primary isotope effects has not chang ed in methanol, and we conclude little or no change in kinetic complexity. Importantly, the degree of tunneling, as shown from the relationship betwee n the secondary k(H)/k(T) and k(D)/k(T) values, is the same in water and Me OH/water mixtures, implicating similar trajectories for H transfer in both solvents. In a recent study of a thermophilic alcohol dehydrogenase [Kohen et al. (1999) Nature 399, 496], it was shown that decreases in temperatures below a transition temperature lead to decreased tunneling. This arises be cause of a change in protein dynamics below a break point in enzyme activit y [Kohen et al. (2000) J. Am. Chem. Sec. 122, 10738-10739]. For the mesophi lic HLADH described herein, an opposite trend is observed in which tunnelin g increases at subzero temperatures. These differences are attributed to in herent differences in tunneling probabilities between 0 and 100 degreesC vs subzero temperatures, as opposed to fundamental differences in protein str ucture for enzymes from mesophilic vs thermophilic sources. We propose that future investigations of the relationship between protein flexibility and hydrogen tunneling are best approached using enzymes from thermophilic sour ces.