Enzyme dynamics and their role in formate dehydrogenase

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.