Purification and mechanism of delta 3,delta 5-t-2,t-4-dienoyl-CoA isomerase from rat liver.
A new enzyme, i.e., delta 3,delta 5-t-2,t-4-dienoyl-CoA isomerase, required in the NADPH-dependent metabolic pathway of odd-numbered double bond, unsaturated fatty acids, was isolated and purified to apparent homogeneity from rat liver. In the oxidation of odd-numbered double bond, unsaturated fatty acids, stepwise beta-oxidation leads to cis-5-enoyl-CoA, which is then dehydrogenated and isomerized to delta 3,delta 5-dienoyl-CoA. delta 3,delta 5-t-2,t-4-Dienoyl-CoA isomerase converts delta 3,delta 5-dienoyl-CoA to trans-2,trans-4-dienoyl-CoA, which is a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase. This enzyme was purified through Matrex gel red A, blue Sepharose, DEAE-cellulose, CM-cellulose, hydroxylapatite, and Sepharose CL6B column chromatography of an ammonium sulfate precipitated fraction (30-80%) of rat liver homogenate. A native molecular weight of 200,000 with four subunits of 55,000 each was determined. The isoelectric point was 6.5. This enzyme was located in mitochondria and was inducible by clofibrate treatment. Using delta 3,delta 5-decadienoyl-CoA, delta 3,delta 5-dodecadienoyl-CoA, and delta 3,delta 5-tetradecadienoyl-CoA as substrates, the Vmax ratio was 1:0.5:0.4 and the Km's were 10.9, 5.9, and 1.4 microM, respectively. The specific activity of purified enzyme was 7 units/mg using delta 3,delta 5-decadienoyl-CoA as substrate. The mechanism of isomerization was studied by deuterium labeling. Consistent with the deuterium labeling pattern of the products, the isomerization from trans-2,cis-5-dienoyl-CoA to trans-2,trans-4-dienoyl-CoA was a two-step process through an intermediate delta 3,delta 5-dienoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
L S
LS
Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio.
Jin
S J
SJ
Tserng
K Y
KY
D000818:Animals; D019748:Carbon-Carbon Double Bond Isomerases; D002994:Clofibrate / Q000494:pharmacology; D003903:Deuterium; D064006:Dodecenoyl-CoA Isomerase; D005215:Fasting / Q000378:metabolism; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011487:Protein Conformation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013237:Stereoisomerism; D013379:Substrate Specificity; D014018:Tissue Distribution
D003903:Deuterium; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C085575:delta(3,5),delta(2,4)-dienoyl-CoA isomerase; D064006:Dodecenoyl-CoA Isomerase; D002994:Clofibrate