PMID:16165092
| Citation |
Kalliri, E, Grzyska, PK and Hausinger, RP (2005) Kinetic and spectroscopic investigation of CoII, NiII, and N-oxalylglycine inhibition of the FeII/alpha-ketoglutarate dioxygenase, TauD. Biochem. Biophys. Res. Commun. 338:191-7 |
|---|---|
| Abstract |
Co(II), Ni(II), and N-oxalylglycine (NOG) are well-known inhibitors of Fe(II)/alpha-ketoglutarate (alphaKG)-dependent hydroxylases, but few studies describe their kinetics and no spectroscopic investigations have been reported. Using taurine/alphaKG dioxygenase (TauD) as a paradigm for this enzyme family, time-dependent inhibition assays showed that Co(II) and Ni(II) follow slow-binding inhibition kinetics. Whereas Ni(II)-substituted TauD was non-chromophoric, spectroscopic studies of the Co(II)-substituted enzyme revealed a six-coordinate site (protein alone or with alphaKG) that became five-coordinate upon taurine addition. The Co(II) spectrum was not perturbed by a series of anions or oxidants, suggesting the Co(II) is inaccessible and could be used to stabilize the protein. NOG competed weakly (Ki approximately 290 microM) with alphaKG for binding to TauD, with the increased electron density of NOG yielding electronic transitions for NOG-Fe(II)-TauD and taurine-NOG-Fe(II)-TauD at 380 nm (epsilon380 90-105 M(-1) cm(-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. |
| Links |
PubMed Online version:10.1016/j.bbrc.2005.08.223 |
| Keywords |
Amino Acid Sequence; Amino Acids, Dicarboxylic/chemistry; Cobalt/chemistry; Enzyme Inhibitors/chemistry; Escherichia coli; Humans; Kinetics; Mixed Function Oxygenases/antagonists & inhibitors; Mixed Function Oxygenases/chemistry; Mixed Function Oxygenases/metabolism; Molecular Sequence Data; Nickel/chemistry; Recombinant Proteins/antagonists & inhibitors; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Spectrophotometry, Ultraviolet |
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