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.
Bornemann
S
S
Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
Balasubramanian
S
S
Coggins
J R
JR
Abell
C
C
Lowe
D J
DJ
Thorneley
R N
RN
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