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°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°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.hydrogen tunneling | enzyme kinetic | secondary isotope effect | Swain-Schaad E nzymes enhance the rates of chemical reactions by many orders of magnitude, and extensive studies have uncovered many aspects of how they do so. The prevailing theory that enzymes stabilize the transition state (TS) relative to the reactants explains many phenomena, and a great deal of contemporary research focuses on how enzymes stabilize the TS. An unfortunate hindrance to developing an understanding of how enzymes stabilize the TS lies in the enigmatic nature of the TS. Many enzymatic hydrogen (proton, hydrogen, or hydride) transfers, for example, occur by quantum mechanical tunneling, where a particle passes through an energy barrier because of its wave properties (1-6).Yeast alcohol dehydrogenase (yADH) serves as an excellent model system for enzyme-catalyzed H transfer because unlike many other enzymes, the chemical step, oxidation of a primary alcohol to an aldehyde by NAD þ , is rate-limiting with aromatic substrates (7). Furthermore, the thermodynamics of the reaction allow for examination of both the forward (alcohol to aldehyde) and reverse (aldehyde to alcohol) reactions under similar conditions (8). This type of nicotinamide-dependent redox reaction appears ubiquitously in biology, and a detailed picture of the TS of such reactions may facilitate many medical and industrial applications.Intriguingly, decades of experiments on the reaction catalyzed by yADH have returned what appear to be contradictory results, some suggesting an early TS, whereas others point to a late TS (7-10). In pioneering studies, Klinman measured linear fr...

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

“…Intrinsic KIEs for labeled inosines as substrates for PNP reflect differences in bond vibrational environment between substrates in solution and at the transition state (24). Accordingly, KIE analysis has been used to probe transition-state structures of PNP as well as several related enzyme-catalyzed reactions (6,(27)(28)(29)(30). The intrinsic KIEs of heavy and light PNP are indistinguishable within experimental error ( Table 1), indicating that altered femtosecond protein dynamic motions do not change the geometry of the PNP transition state.…”

Isotope Effects as Probes for Enzyme Catalyzed Hydrogen-Transfer Reactions

“…([40] did not appear so far.) In some cases, the observed isotope effect on the pre-exponential factoris greater than unity, corresponding to smaller than expected temperature dependence of KIEs, whereas other reactions exhibit an isotope effect on the pre-exponential factor that is smaller than unity, corresponding to larger than expected temperature dependence.…”

The geochemistry of methane isotopologues

“…2.7); this is the main source of uncertainty in estimates of CH 4 (g)/H 2 O(l) equilibrium D/H fractionation, which is derived by combination of H 2 O(l)/H 2 (g), H 2 (g)/H 2 O(g), and CH 4 (g)/H 2 (g) calibration curves. We used the Cerrai et al (1954) calibration for H 2 O(l)/ H 2 (g) in the calculation of ε methane/water of the equilibrium endmember of our model for isotope effects accompanying microbial methanogenesis (see § 2.4.7) because amongst the published calibrations, this is likely most accurate at lower temperatures (Suess, 1949;Horibe and Craig, 1995;Roston and Kohen, 2010). The uncertainty in calibration, as well as salt and pressure effects (Horita, 2005), could explain small apparent offsets from the equilibrium line ( Fig.…”

“…For example for reservoir D, the continuity equation for the D-substituted isotopologue (i.e., R=CH 2 or R=CHD) is: Table 2.3). The intrinsic (kinetic/forward) 13 C/ 12 C and D/H fractionation factors are estimated from in vitro and culture studies (Hermes et al, 1984;Scharschmidt et al, 1984;Valentine et al, 2004;Roston and Kohen, 2010;Scheller et al, 2013 (Cerrai et al, 1954;Horibe and Craig, 1995;Horita, 2001;Ono et al, 2014). The intrinsic fractionation factors for the reverse reactions (α − , Table 2.3) are constrained by the requirement for consistency among equilibrium (α eq ), forward (α + ), and reverse reactions (i.e., α eq = α − /α + ).…”

“…For simplicity, primary (α p ) and secondary (α s ) kinetic isotope fractionation factors for the four Haddition steps are assumed to be identical at a given temperature (fractionation factors calculated for a model temperature of 20°C are shown in Table 2.3). The intrinsic (kinetic/forward) 13 C/ 12 C and D/H fractionation factors are estimated from in vitro and culture studies (Hermes et al, 1984;Scharschmidt et al, 1984;Valentine et al, 2004;Roston and Kohen, 2010;Scheller et al, 2013). The intrinsic 13 CD fractionation factor (γ, where (Cerrai et al, 1954;Horibe and Craig, 1995;Horita, 2001;Ono et al, 2014).…”