Stopped-Flow Kinetic Analysis ofEscherichia coliTaurine/α-Ketoglutarate Dioxygenase: Interactions with α-Ketoglutarate, Taurine, and Oxygen

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Abstract
Taurine/α-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde while decomposing α-ketoglutarate (αKG) to form succinate and CO2. Under anaerobic conditions, the addition of αKG to Fe(II)TauD results in the formation of a broad absorption centered at 530 nm. On the basis of studies of other members of the αKG-dependent dioxygenase superfamily, we attribute this spectrum to metal chelation by the substrate C-1 carboxylate and C-2 carbonyl groups. Subsequent addition 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-aminoethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), αKG, and taurine. In addition, these studies indicate that the affinity of TauD for αKG is enhanced by the presence of taurine. α-Ketoadipate, the other α-keto acid previously shown to support TauD activity, and α-ketocaproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD in the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and α-ketoisovalerate fail to elicit absorption bands in this region of the spectrum, even in the presence of taurine. Stopped-flow UV−visible spectroscopy reveals that the 530 and 520 nm spectra associated with αKG−Fe(II)TauD and taurine−αKG−Fe(II)TauD are formed at catalytically competent rates (∼40 s-1). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that αKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine−αKG−Fe(II)TauD state, but not the αKG−Fe(II)TauD species, reacts rapidly with oxygen (42 ± 9 s-1). Using the data described herein, we develop a preliminary kinetic model for TauD catalysis.