PMID:10563813
| Citation |
Ryle, MJ, Padmakumar, R and Hausinger, RP (1999) Stopped-flow kinetic analysis of Escherichia coli taurine/alpha-ketoglutarate dioxygenase: interactions with alpha-ketoglutarate, taurine, and oxygen. Biochemistry 38:15278-86 |
|---|---|
| Abstract |
Taurine/alpha-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 alpha-ketoglutarate (alphaKG) to form succinate and CO(2). Under anaerobic conditions, the addition of alphaKG 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 alphaKG-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), alphaKG, and taurine. In addition, these studies indicate that the affinity of TauD for alphaKG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-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 alpha-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 alphaKG-Fe(II)TauD and taurine-alphaKG-Fe(II)TauD are formed at catalytically competent rates ( approximately 40 s(-)(1)). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that alphaKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine-alphaKG-Fe(II)TauD state, but not the alphaKG-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. |
| Links | |
| Keywords |
Binding Sites; Escherichia coli/enzymology; Ferrous Compounds/metabolism; Ketoglutaric Acids/chemistry; Ketoglutaric Acids/metabolism; Kinetics; Mixed Function Oxygenases/chemistry; Mixed Function Oxygenases/metabolism; Oxygen/chemistry; Oxygen/metabolism; Oxygenases/chemistry; Oxygenases/isolation & purification; Oxygenases/metabolism; Spectrophotometry; Spectrophotometry, Ultraviolet; Substrate Specificity; Taurine/chemistry; Taurine/metabolism |
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