Hydrogen Tunneling in Enzyme Reactions

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9507018 Klinman The original observations of room-temperature hydrogen tunneling in enzyme reactions occured in the yeast alcohol dehydrogenase and the bovine serum amine oxidase reactions. In recent years, evidence for tunneling has been exteneded to include horse liver alcohol dehudrogenase, monoamine oxidase, and very likely soybean lipoxygenase and glucose oxidase. When conditions are optimized to allow the monitoring of the hydrogen transfer step itself, it appears that the phenomenon of tunneling can be readily detected, although the exact nature of tunneling varies from one system to another. Our findings suggest that, in conjuction with the more classical sources of catalytic rate enhancement, the structures at enzyme active sites have been optimized to make catalytic use of quantum effects. One of the most extreme types of behavior has been seen in the soybean lipoxgenase reaction, which is postulated to catalyze substrate oxidation by close to full quantum event. Future studies with this enzyme will focus on (i) the use of stopped flow kinetics to monitor the H-transfer step directly as function of temperature; (ii) the kinetic characterization of D/T and H/T labeled substrates as probes of tunneling. Premiliary studies of D/T and H/T isotope effects with two forms of glucose oxidase, which differ ca. two-fold in mass (due solely to differences in patterns of glycosylation),suggest different degrees of tunneling. These proteins will be deglycosylated and recharacterized to test the hypothesis that overall protein mass (and possibly mobility) can influence tnneling. The horse liver alcohol dehydrogenase offers a superb opportunity to examine the role of specific active site side chains in optimizing tunneling. A systematic study of residues contacting the bound cofactor is planned by site specific mutagenesis. In order or pursue the question of a possible link between protein dynamics and efficient hydrogen tunneling, two experimental routes wi ll be taken. These involve (I) the study of horse liver alcohol dehydrogenase at low temperature in cryosolvent using a photodisociable, substrate precursor and (ii) the study of D/T and H/T isotope effects as a function of temperture (between ca. 25 and 90oC) using thermophilic forms of alcohol dehydrogenase. Brief Description (200 words) of Project The accepted paradigm for the enormous rate accelerations brought about by protein catalysts (enzymes) has been a reduction in the height of the energy barrier for the conversion of reactant to product. Quantum mechanics provides a more fundamental and general way of formalizing reaction dyamics. However, with the exception of electron transfer reactions, it has generally been assumed that proteins, because of their large size, catalyze reactions classically. In fact, we have recently shown that under physiologic conditions, quantum effects contribute significantly to the tate accelerations brought about by many enzymes. We are now trying to understand the specific ways in which enzymes take advantage of quantum mechanics to enhance their catalytic effects. Three areas are under investigation: these include roles of specific amino acid side chain interactions, overall protein size, and protein dynamics in the manifestation of catalysis through quantum effects.