title: The mechanism of dienoyl-CoA reduction by 2,4-dienoyl-CoA reductase is stepwise: observation of a dienolate intermediate.
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 NADPH-dependent reduction of 2,4-dienoyl-coenzyme A (CoA) thiolesters to the resulting trans-3-enoyl-CoA. Steady-state kinetic parameters for trans-2,trans-4-hexadienoyl-CoA and 5-phenyl-trans-2,trans-4-pentadienoyl-CoA were determined and demonstrated that the dienoyl-CoA and NADPH bind to the 2,4-dienoyl-CoA reductase via a sequential kinetic mechanism. Kinetic isotope effect studies and the transient kinetics of substrate binding support a random order of nucleotide and dienoyl-CoA addition. The large normal solvent isotope effects on V/K ((D)(2)(O)V/K) and V ((D)(2)(O)V) for trans-2,trans-4-hexadienoyl-CoA reduction indicate that a proton transfer step is rate limiting for this substrate. The stability gained by conjugating the phenyl ring to the diene in PPD-CoA results in the reversal of the rate-determining step, as evidenced by the normal isotope effects on V/K(CoA) ((D)V/K(CoA)) and V/K(NADPH) ((D)V/K(NADPH)). The reversal of the rate-determining step was supported by transient kinetics where a burst was observed for the reduction of trans-2,trans-4-hexadienoyl-CoA but not for 5-phenyl-trans-2,trans-4-pentadienoyl-CoA reduction. The chemical mechanism is stepwise where hydride transfer from NADPH occurs followed by protonation of the observable dienolate intermediate, which has an absorbance maximum at 286 nm. The exchange of the C alpha protons 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 catalyzed by the enzyme active site.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Fillgrove
authors.forename: V E
authors.initials: VE
authors.lastname: Anderson
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D003903:Deuterium; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011522:Protons; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents; D013379:Substrate Specificity
chemical_list: C090296:5-phenyl-2,4-pentadienoyl-coenzyme A; D000214:Acyl Coenzyme A; D011522:Protons; D011994:Recombinant Proteins; D012997:Solvents; C417225:hexadienoyl-coenzyme A; D009249:NADP; C040351:2,4-decadienoyl-coenzyme A; D003903:Deuterium; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase