The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: Observation of a dienolate intermediate

Citation
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
Citations number
55
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
40
Issue
41
Year of publication
2001
Pages
12412 - 12421
Database
ISI
SICI code
0006-2960(20011016)40:41<12412:TMODRB>2.0.ZU;2-D
Abstract
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.