An Active-Site Phenylalanine Directs Substrate Binding and C-H Cleavage in the alpha-Ketoglutarate-Dependent Dioxygenase TauD

作者：McCusker Kevin P; Klinman Judith P^*

来源：Journal of the American Chemical Society, 2010, 132(14): 5114-5120.

DOI：10.1021/ja909416z

Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine a-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

• 出版日期2010-4-14