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) 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.
Ryle
M J
MJ
Department of Microbiology, Michigan State University, East Lansing 48824-1011, USA.
Padmakumar
R
R
Hausinger
R P
RP
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005296:Ferrous Compounds / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013654:Taurine / Q000737:chemistry* / Q000378:metabolism
D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D013654:Taurine; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D010100:Oxygen