Elusive transition state of alcohol dehydrogenase unveiled

Abstract

For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2 degrees kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2 degrees KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.

Fig. 1.

Schematic TRS of the reverse reaction (aldehyde to alcohol) showing 1°–2° coupling. The hydrogens are black, the carbons are gray, the oxygen is red, and the three heavy atoms define the blue plane. Traditional models of tunneling and coupled motion proposed that the reaction coordinate involved motion of all three hydrogens (shown with arrows). The model presented here parameterized the out-of-plane bending angles of the benzyl substrate and nicotinamide cofactor, θ_s and θ_c, respectively, in order to obtain a symmetric double-well potential along the H-transfer coordinate.

Fig. 2.

Marcus-like model of a reaction with H tunneling. At the TRS (‡), the reactant (Black) and product (Gray) surfaces are degenerate, which allows the probability density of the hydrogen (Shaded) to spread from the donor well to the acceptor well (i.e., quantum mechanical tunneling) at a rate dependent on the DAD and the particle’s mass. Once degeneracy is broken, the hydride wave function can collapse into the acceptor well, giving a net transfer (i.e., dissipative tunneling). The Marcus coordinate is a complex amalgamation of many modes, some of the most important of which are discussed in the text.

Fig. 3.

Least-squares quartic fit to 25 single point calculations at the B3LYP/6-31 + G^∗ level along the H-transfer coordinate at the TRS. The vibrational wave functions of the ground state (Dashed Line) and first excited state (Dashed-Dotted Line) of the tunneling hydride were calculated as described in SI Text. This surface is equivalent to the 1D slice of the “hydride coordinate” at the TRS in Fig. 2.

Fig. 4.

TRS structure for H transfer found by the present methods, showing all heavy atoms used in the model along with hydrogen atoms of particular interest. The dividing surface of conformations that yield a TRS with two degenerate wells (ΔE_D-A = 0) is quite broad, so this structure represents the weighted average (via Boltzmann distribution) of the full conformational ensemble for which ΔE_D-A = 0. The 1° hydrogen is shown in both the donor and acceptor positions, but faded, because it has just half of its probability density in each position. Important geometric values are listed in Table 2.

Fig. 5.

Rehybridization during the course of the reaction. The location of the TRS is marked (‡) and the dashed line indicates a reasonable reaction pathway from reactants to products that passes through the TRS. The solid line indicates a reaction pathway with perfectly synchronized rehybridization. The dotted line indicates the surface with perfectly symmetric rehybridization.

Fig. 6.

The effect of para substituents on the forward and reverse reactions catalyzed by yADH. In accordance with previous studies (7, 8) the experimental rates (Dotted Line) (10) showed that the forward reaction is unaffected by electronic changes (substituents with different values of σ⁺) but that electron-withdrawing substituents greatly accelerate the reverse reaction. Calculations (Dashed Line) of the reaction with a representative range of para substituents (-OCH₃,-H,-Br) showed similar trends in the change of Mulliken charge (ΔQ_M) on the benzylic carbon between reactants and the TRS. Substituents on benzyl alcohol (forward) do not affect the electronic changes along the reaction coordinate, but substituents on benzaldehyde (reverse) severely alter the electronic changes that accompany the TRS.