Escherichia coli chorismate synthase: a deuterium kinetic-isotope effect under single-turnover and steady-state conditions shows that a flavin intermediate forms before the C-(6proR)-H bond is cleaved.
We report the observation of a deuterium kinetic isotope effect for the conversion of 5-enolpyruvylshikimate-3-phosphate into chorismate (6proR2HV = 1.13 +/- 0.03) using recombinant chorismate synthase from Escherichia coli. Similar isotope effects were observed for the decay of a spectroscopically characterized flavin intermediate (6proR2Hk = 1.17 +/- 0.04) during single-turnover experiments. The main rate-limiting steps and C-(6proR)-H bond breaking are therefore distinct and both must occur after the formation of the flavin intermediate and either before or concomitant with its decay.
Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
S
S
Bornemann
S
S
Balasubramanian
J R
JR
Coggins
C
C
Abell
D J
DJ
Lowe
R N
RN
Thorneley
D001665:Binding Sites; D002827:Chorismic Acid / Q000378:metabolism; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D005415:Flavins / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases; D011994:Recombinant Proteins / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D013053:Spectrophotometry
D005415:Flavins; D011994:Recombinant Proteins; C058070:5-enolpyruvoylshikimate-3-phosphate; D012765:Shikimic Acid; D003903:Deuterium; D008190:Lyases; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases; D002827:Chorismic Acid