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

摘要

Abstract: Enzymes that cleave C H bonds are often found to depend on well-packed hydrophobic coresnthat influence the distance between the hydrogen donor and acceptor. Residue F159 in taurinen-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientationnof its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)dO. Mutation of F159nto smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding andnfor C H bond cleavage, as well as increased contribution of the chemical step to kcat under steady-statenturnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at positionn159 than observed for the oxygen activation process supports a previous conclusion of modularity of functionnwithin the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,n19791 19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transientsnshows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appearsnthat there is a break in behavior following removal of a single methylene from the side chain of F159L tongenerate F159V, whereby the protein active site loses its ability to restore the internuclear distance betweennsubstrate and Fe(IV)dO that supports optimal hydrogenic wave function overlap.