Stopped-flow kinetic analysis of Escherichia coli taurine/alpha-ketoglutarate dioxygenase: Interactions with alpha-ketoglutarate, taurine, and oxygen

Taurine/alpha-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) t o sulfite and aminoacetaldehyde while decomposing alpha-ketoglutarate (alph a KG) to form succinate and CO2. Under anaerobic conditions, the addition o f alpha KG to Fe(II)TauD results in the formation of a broad absorption cen tered at 530 nm. On the basis of studies of other members of the alpha KG-d ependent dioxygenase superfamily, we attribute this spectrum to metal chela tion by the substrate C-l carboxylate and C-2 carbonyl groups. Subsequent a ddition of taurine perturbs the spectrum to yield a 28% greater intensity, an absorption maximum at 520 nm, and distinct shoulders at 480 and 570 nm. This spectral change is specific to taurine and does not occur when 2-amino ethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), alpha KG, and ta urine. In addition, these studies indicate that the affinity of TauD for al pha KG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-ketoc aproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD i n the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and alpha-ketoisovalerate fai l to elicit absorption bands in this region of the spectrum, even in the pr esence of taurine. Stopped-flow UV-visible spectroscopy reveals that the 53 0 and 520 nm spectra associated with alpha KG-Fe(II)TauD and taurine-alpha KG-Fe(II)TauD are formed at catalytically competent rates (similar to 40 s( -1)). The rate of chromophore formation was independent of substrate or enz yme concentration, suggesting that aKG binds to Fe(II)TauD prior to the for mation of a chromophoric species. Significantly, the taurine-alpha KG-Fe(II )TauD state, but not the alpha KG-Fe(II)TauD species, reacts rapidly with o xygen (42 +/- 9 s(-1)). Using the data described herein, we develop a preli minary kinetic model for TauD catalysis.