ISOLATION AND CHARACTERIZATION OF RAT-LIVER MICROSOMAL R-IBUPROFENOYL-COA SYNTHETASE

Microsomal long-chain acyl-CoA synthetase (EC 6.1.2.3.) has been sugge sted to be involved in the stereoselective formation of the CoA thioes ter of ibuprofen. In this study, we demonstrated that the microsomal e nzyme from rat liver responsible for palmitoyl-CoQI synthesis also cat alyzes the formation of R-ibuprofenoyl-CoA in a Mg2+- and ATP-dependen t process. Long-chain acyl-CoA synthetase from rat liver microsomes wa s purified to homogeneity as evidenced by SDS-gel electrophoresis. Sim ultaneous measurements of palmitoyl-CoA and R-ibuprofenoyl-CoA formati on with HPLC in various fractions and purification steps during protei n isolation revealed a high correlation between both activities. The p urification procedure included solubilization of the microsomes obtain ed from rat livers with Triton X-100 and subsequent chromatography of the 100,000 x g supernatant on blue-sepharose, hydroxyapatite, and pho sphocellulose. The purified enzyme exhibited an apparent molecular wei ght of 72 kDa as estimated by SDS gel electrophoresis, with specific a ctivities of 71 nmol . min(-1). mg(-1) protein and 901 nmol . min(-1). mg(-1) protein for formation of R-ibuprofenoyl-CoA and palmitoyl-CoA, respectively. Palmitoyl-CoA formation catalyzed by the purified enzym e exhibited biphasic kinetics indicative of two isoforms, a high-affin ity (K-M 0.13 +/- 0.11 mu M), low-capacity form and a low-affinity (K- M 81 +/- 11.5 mu M), high-capacity form. In contrast, measurement of R -ibuprofenoyl-CoA synthesis over a concentration range from 5 to 3000 mu M showed the participation of a single CoA ligase with a K-M of 184 +/- 19 mu M, corresponding to the low-affinity isoform of palmitoyl-C oA synthesis with a marked enantioselectivity towards the R-form of ib uprofen. R-ibuprofenoyl-CoA formation of the enzyme preparation was in hibited by palmitic acid (K-I 13.5 +/- 0.5 mu M) and S-ibuprofen (K-I 405 +/- 10 mu M). In summary, these data give strong evidence for the identity of R-ibuprofenoyl-CoA and long-chain acyl-CoA synthetase.