SPECIFIC INTERACTION OF (R)-3-HYDROXYBUTYRATE DEHYDROGENASE WITH MEMBRANE PHOSPHATIDYLCHOLINE AS STUDIED BY ESR SPECTROSCOPY IN ORIENTED PHOSPHOLIPID MULTIBILAYERS - COENZYME BINDING ENHANCES THE INTERACTION WITH PHOSPHATIDYLCHOLINE

The interaction of phospholipid with (R)-3-hydroxybutyrate dehydrogena se, a phosphatidylcholine-requiring membrane enzyme, has been studied using ESR spectroscopy of spin-labeled lipids, both as ordered multibi layers and in lipid vesicle suspensions (liposomes). Partially oriente d phospholipid multibilayers were prepared from lipid vesicles compose d of a 1:1 mixture of phosphatidylcholine (PC) and phosphatidylethanol amine (PE), Vesicles containing (R)-3-hydroxybutyrate dehydrogenase yi elded active preparations of the enzyme in such multibilayers. With in creasing protein/lipid ratio. the older of the multibilayers was disru pted as monitored by ESR spectroscopy with a spin-labeled analogue of PC, 5-doxyl-PC (5 mol %, 10% of total PC) as a probe. The outer peak s eparation of 5-doxyl-PC varied with the lipid/protein ratio, The lower the ratio, the larger was the separation, with higher activity enzyme being more effective in exerting this effect. When 5-doxylstearic aci d was substituted for 5-doxyl-PC or when the enzyme was inactive, the 2A(zz) value stayed practically constant at its lower limit (about 54 G), Multilayers composed of 81 % PE, 11% diphosphatidylglycerol (DPG), and 8 % 5-doxyl-PC (no unlabeled PC present) gave similar results. Wi th this lipid mixture, the maximal 2A(zz) value (about 61 G) was reach ed at lower protein/lipid ratios, although the enzymic activity of (R) -3-hydroxybutyrate dehydrogenase is reduced to 40% in this system,The outer peak separation also depended on the presence of the coenzyme, N AD(+), and 2-methylmalonate. The latter enhances binding of NAD(+) abo ut 100-fold by farming a ternary complex, With this ternary complex, t he 2A(zz) values were increased unless the maximal values had been rea ched already in the absence of coenzyme. In all these experiments only a single ESR spectral component was observed. Similar results were ob tained for the enzyme in liposomes, although the effect was less prono unced apparently due to the higher mobility of the probe. It is conclu ded that PC is motionally restricted by (R)-3-hydroxybutyrate dehydrog enase and yet is in rapid exchange with the bulk lipid on the ESR time scale. PC is required for formation of tight and functional complexes with NAD [Rudy et al. (1989) Biochemistry 28, 5354-5366], and such co mplexes strengthen the interaction of the enzyme with PC.