Kinetic and spectroscopic investigation of Co{sup II}, Ni{sup II}, and N-oxalylglycine inhibition of the Fe{sup II}/{alpha}-ketoglutarate dioxygenase, TauD

Abstract

Co{sup II}, Ni{sup II}, and N-oxalylglycine (NOG) are well-known inhibitors of Fe{sup II}/{alpha}-ketoglutarate ({alpha}KG)-dependent hydroxylases, but few studies describe their kinetics and no spectroscopic investigations have been reported. Using taurine/{alpha}KG dioxygenase (TauD) as a paradigm for this enzyme family, time-dependent inhibition assays showed that Co{sup II} and Ni{sup II} follow slow-binding inhibition kinetics. Whereas Ni{sup II}-substituted TauD was non-chromophoric, spectroscopic studies of the Co{sup II}-substituted enzyme revealed a six-coordinate site (protein alone or with {alpha}KG) that became five-coordinate upon taurine addition. The Co{sup II} spectrum was not perturbed by a series of anions or oxidants, suggesting the Co{sup II} is inaccessible and could be used to stabilize the protein. NOG competed weakly (K {sub i} {approx} 290 {mu}M) with {alpha}KG for binding to TauD, with the increased electron density of NOG yielding electronic transitions for NOG-Fe{sup II}-TauD and taurine-NOG-Fe{sup II}-TauD at 380 nm ({epsilon} {sub 38} 90-105 M{sup -1} cm{sup -1}). The spectra of the NOG-bound TauD species did not change significantly upon oxygen exposure, arguing against the formation of an oxygen-bound state mimicking an early intermediate in catalysis.