TY - JOUR
T1 - Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase
AU - Stahl, Shannon S.
AU - Francisco, Wilson A.
AU - Merkx, Maarten
AU - Klinman, Judith P.
AU - Lippard, Stephen J.
PY - 2001/2/16
Y1 - 2001/2/16
N2 - Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH3CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH2CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 ± 0.0007 and 1.0167 ± 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turn-over and steady-state kinetic studies.
AB - Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH3CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH2CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 ± 0.0007 and 1.0167 ± 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turn-over and steady-state kinetic studies.
UR - http://www.scopus.com/inward/record.url?scp=0035895924&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035895924&partnerID=8YFLogxK
U2 - 10.1074/jbc.M008301200
DO - 10.1074/jbc.M008301200
M3 - Article
C2 - 11073959
AN - SCOPUS:0035895924
SN - 0021-9258
VL - 276
SP - 4549
EP - 4553
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 7
ER -