Enzymes use a number of common cofactors as sources of hydrogen
Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes but the physics of the hydrogen transfers to and from these cofactors is not fully understood. of two case studies. The first example is alcohol dehydrogenase which uses a nicotinamide cofactor to catalyze a hydride transfer and the second example is usually thymidylate synthase which uses a folate cofactor to catalyze both a hydride and a proton transfer. SSEs in ADH for the mixed labeling experiment but no deviations have been reported for any measurement of real SSE (in contrast to mixed SSE as discussed below). In the sections that follow we will discuss two case studies: the nicotinamide in ADH and the folate PAC-1 in TSase. In ADH the Northrop method has been largely unnecessary but in many mutants of TSase the Northrop method has been instrumental in extracting KIEint. 3 Nicotinamide in Alcohol Dehydrogenase Nicotinamide cofactors such as nicotinamide adenine dinucleotide (NADH) or its 2’-phosphate analogue (NADPH) are ubiquitous in biology and ADH provides a very useful model system for studying the physical mechanism of nicotinamide-dependent hydride transfers. ADH catalyzes the oxidation of alcohol shown in plan 1 and is a particularly useful model because in the yeast enzyme (yADH) the hydride transfer is completely exposed so physical measurements are not hindered by kinetic complexity . Furthermore the reaction can proceed in both the forward and reverse directions using relatively similar conditions [37 42 Much of the work on ADH has focused on using KIEs especially 2° KIEs to understand the nature of the TS and the roll of tunneling and dynamics in H-transfers. The amazing results of many of these KIE experiments though have been difficult to connect to a demanding theoretical framework. Plan 1 The hydride transfer catalyzed by ADH using benzyl alcohol as an alternative substrate. R= adenine diphosphate ribosyl. Some of the first difficulties appeared when Klinman and coworkers compared 2° KIEs with S1PR2 Hammett substituent effects [37 42 43 The α-2° KIE on alcohol oxidation was very close to the corresponding EIE indicating a very late (product-like) TS  whereas the substituent effects indicated just the opposite; the electronic structure of the TS was very reactant-like [37 42 Clearly this kind of blatant contradiction could not be rationalized by traditional semi-classical theories and was perhaps the first indication that those theories were missing a vital component to the mechanism of H-transfer. Shortly after these experiments Cleland and coworkers probed the reaction by measuring isotope effects around the cofactor and obtained some remarkable results [31 44 Despite the fact that the relevant EIE was inverse as expected for the sp2 to sp3 transition of the cofactor in the backward reaction the measured KIE was significantly normal. The authors proposed that this startling result indicated a component of 1°-2° coupled motion (Physique 3) in the reaction coordinate. Some theoretical calculations  verified the validity of that interpretation but added to it the fact that the coupled reaction coordinate mode tunneled through the barrier. This theoretical model further predicted that tunneling and coupled motion would lead to a breakdown of the rule of the geometric imply (RGM). The RGM is usually a consequence of the semi-classical Bigeleisen model and says that PAC-1 there are no isotope effects on isotope effects [45 46 After the theoretical model suggested that tunneling and coupled motion would lead to a breakdown Cleland and coworkers tested the RGM in formate dehydrogenase which also uses NAD+ as an oxidizing agent. The experiments found that indeed the RGM failed to hold in this reaction providing strong evidence for tunneling and coupled motion in nicotinamide-dependent H-transfers. Physique 3 Schematic of the model of tunneling and coupled motion. A) The reaction coordinate is composed of motion of 3 hydrogens (black): the transferred atom moving from donor to acceptor carbon (gray) and PAC-1 the 2° hydrogens swinging around as the donor … PAC-1 Another important prediction  of the tunneling and coupled motion model was that the Swain-Schaad exponent (SSE) would be inflated from its semi-classical.