The Exit of the Tunnel: Yeast Alcohol Dehydrogenase from the Acceptor's Point of View

Abstract

Alcohol dehydrogenase (ADH) is a popular model used to study quantum mechanical phenomena in enzyme-catalyzed reactions. Studies of α-secondary kinetic isotope effects (2° KIEs) have shown that the oxidation of benzyl alcohol by NAD+ occurs by quantum tunneling with coupled motion of the primary and secondary hydrogens. In order to learn more about the nature of that coupling, we have measured α-secondary KIEs in the reverse reaction, i.e., for the yeast ADH (yADH) catalyzed reduction of benzaldehyde to benzyl alcohol. Preliminary results show that whether 1H or 2H is being transferred, the reaction maintains normal 2° KIEs (kH/kT > 1). This is most significant given that the equilibrium isotope effect for this process is inverse (EIE = 0.75). Semi-classical theory predicts an inverse 2° KIE (kH/kT < 1) for this reaction, thus the findings support a role for quantum mechanical H-tunneling in the reduction of aldehydes by yADH. Furthermore, these 2° KIEs violate the rule of the geometric mean: the semi-classical formulation of KIEs that predicts no difference in 2° KIEs upon isotopic substitution at the primary position. In this reaction, however, the magnitude of the 2° KIE decreases significantly when the transferred isotope is 2H. Together, these results provide strong support for the model of tunneling and 1°-2° coupled motion used to describe enzymatic H-transfers.