Solution and Computational Studies of Kinetic Isotope Effects in Flavoprotein and Quinoprotein Catalyzed Substrate Oxidations as Probes of Enzymic Hydrogen Tunneling and Mechanism

I. Enzymic H-Tunneling and Kinetic Isotope Effects ........................................................ 671

A. Stopped-Flow Methods to Access the Half-Reactions

of Flavoenzymes and Quinoproteins ....................................................................... 672

II. Interpreting Temperature Dependence of Isotope Effects in Terms

of H-Tunneling................................................................................................................. 673

III. H-Tunneling in Flavoenzymes PETN Reductase and MR ............................................. 675

IV. H-Tunneling in TTQ-Dependent MADH and AADH .................................................... 678

V. Computational Studies of Substrate Oxidation in TTQ-Dependent

Amine Dehydrogenases ................................................................................................... 679

VI. H-Tunneling in Flavoprotein Amine Dehydrogenases: TSOX and

Engineering Gated Motion in TMADH .......................................................................... 682

VII. Concluding Remarks........................................................................................................ 685

Acknowledgments ........................................................................................................................ 685

References..................................................................................................................................... 685

Kinetic isotope effects (KIEs) are powerful probes of H-transfer reactions and have provided

evidence for nonclassical transfer of the H nucleus in enzymes (see Chapter 28 by Kohen in this

volume for a detailed discussion of the use of KIEs to identify tunneling regimes). Early

studies of H-transfer by quantum tunneling focused on deviations from values predicted by

semi-classical models (in which zero point energies, but not tunneling, have been taken into

account): KIEs, Swain-Schaad relationships

[exp

. 3:26; where k

, k

, and k

are the

rates of transfer for protium, deuterium and tritium, respectively] or Arrhenius prefactor ratios

(q1 for a reaction proceeding purely by tunneling, ,1 for moderate tunneling). For a more

detailed discussion see, for example, Chapter 28 by Kohen in this volume. Early examples in

which H-tunneling was inferred from measurements of KIEs include yeast alcohol

dehydrogenase,

bovine serum amine oxidase,

horse liver alcohol dehydrogenase,

and

monoamine oxidase.