Kl. Fillgrove et Ve. Anderson, The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: Observation of a dienolate intermediate, BIOCHEM, 40(41), 2001, pp. 12412-12421
The chemical mechanism of the 2,4-dienoyl-CoA reductase (EC 1.3.1.34) from
rat liver mitochondria has been investigated. This enzyme catalyzes the NAD
PH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the r
esulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,tra
ns-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were det
ermined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-die
noyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effe
ct studies and the transient kinetics of substrate binding support a random
order of nucleotide and dienoyl-CoA addition. The large normal solvent iso
tope effects on V/K (V-D2O/K) and V (V-D2O) for trans-2,trans-4-hexadienoyl
-CoA reduction indicate that a proton transfer step is rate limiting for th
is substrate. The stability gained by conjugating, the phenyl ring to the d
iene in PPD-CoA results in the reversal of the rate-determining step, as ev
idenced by the normal isotope effects on V/K-CoA (V-D/K-CoA) and V/K-NADPH
(V-D/K-NADPH). The reversal of the rate-determining step was supported by t
ransient kinetics where a burst was observed for the reduction of trans-2,t
rans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-Co
A reduction. The chemical mechanism is stepwise where hydride transfer from
NADPH occurs followed by protonation of the observable dienolate intermedi
ate, which has an absorbance maximum at 286 nm. The exchange of the Ca prot
ons of trans-3-decenoyl-CoA, catalyzed by the 2,4-dienoyl-CoA reductase, in
the presence of NADP(+) suggests that formation of the dienolate is cataly
zed by the enzyme active site.