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, r equired in the NADPH-dependent metabolic pathway of odd-numbered doubl e bond, unsaturated fatty acids, was isolated and purified to apparent homogeneity from rat liver. In the oxidation of odd-numbered double b ond, unsaturated fatty acids, stepwise beta-oxidation leads to cis-5-e noyl-CoA, which is then dehydrogenated and isomerized to Delta(3),Delt a(5)-dienoyl-CoA. Delta(3),Delta(5)-t-2,t-4-Dienoyl-CoA isomerase conv erts Delta(3),Delta(5)-dienoyl-CoA to trans-2,trans-4-dienoyl-CoA, whi ch is a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase. This enzyme was purified through Matrex gel red A, blue Sepharose, DEAE-cel lulose, CM-cellulose, hydroxylapatite, and Sepharose CL6B column chrom atography of an ammonium sulfate precipitated fraction (30-80%) of rat liver homogenate. A native molecular weight of 200 000 with four subu nits of 55 000 each was determined. The isoelectric point was 6.5. Thi s enzyme was located in mitochondria and was inducible by clofibrate t reatment. Using Delta(3),Delta(5)-decadienoyl-CoA, Delta(3),Delta(5)-d odecadienoyl-CoA, and Delta(3),Delta(5)-tetradecadienoyl-CoA as substr ates, the V-max ratio was 1:0.5:0.4 and the K-m's were 10.9, 5.9, and 1.4 mu M, respectively. The specific activity of purified enzyme was 7 units/mg using Delta(3),Delta(5)-decadienoyl-CoA as substrate. The me chanism of isomerization was studied by deuterium labeling. Consistent with the deuterium labeling pattern of the products, the isomerizatio n 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 . The contribution of this reductive pathway to the overall oxidation of odd-numbered double bond, unsaturated fatty acids is not known, but it is likely to be a major pathway since the dehydrogenation step is also accompanied by isomerization. Once isomerized to Delta(3),Delta(5 )-dienoyl-CoA, the substrate is committed to the reduction pathway.