FATTY-ACIDS AND ANIONIC PHOSPHOLIPIDS ALTER THE PALMITOYL COENZYME-A KINETICS OF HEPATIC MONOACYLGLYCEROL ACYLTRANSFERASE IN TRITON X-100 MIXED MICELLES

In order to gain a better understanding of the kinetics of activation and inhibition of hepatic monoacylglycerol acyltransferase (MGAT) (EC 2.3.1.22) by fatty acid, we examined the effect of fatty acid with res pect to MGAT's long-chain acyl-CoA substrate in Triton X-100 mixed mic elles, At concentrations between 2.5 and 5.3 mol %, oleic acid stimula ted MGAT activity 2-fold, whereas oleic acid inhibited MGAT at concent rations higher than 7.5 mol %. The dependence on palmitoyl-CoA was hig hly cooperative with a Hill constant of greater than 2.4. When present at less than 3 mol %, oleic acid eliminated the lag in the dependence curve. When concentrations of oleic acid were higher than 3 mol % Mic haelis-Menton kinetics were observed with an apparent K-m value of abo ut 54 mu M for palmitoyl-CoA but with progressively decreasing V-max v alues, This effect was not observed with octanoic acid, suggesting tha t the medium-chain fatty acid is unable to associate stably with the m ixed micelle and, thus, cannot substantially alter substrate affinity. When anionic phospholipids were tested, phosphatidic acid, lysophosph atidic acid, phosphatidylserine, and phosphatidylinositol eliminated s ome of the lag in activation by palmitoyl-CoA. At high molar concentra tions of the anionic lipid activators, apparent K-m values ranged from 77 mu M for phosphatidic acid to 196 mu M for phosphatidylinositol. Z witterionic phospholipids had no effect, nor did the non-phospholipid activators bovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but n ot neomycin or KCl, could overcome the inhibitory effect of oleic acid ; thus, the inhibitory effect of fatty acid did not appear to occur by electrostatic interactions, These blockers did not change the effects observed with the anionic phospholipid activators or with the inhibit or, sphingosine. An altered K-m for palmitoyl-CoA in the presence of f atty acid or anionic phospholipid suggests that both long-chain fatty acids and phospholipid cofactors may induce a conformational change in MGAT, thereby altering the enzyme's affinity for its long-chain acyl- CoA substrate, These data further support the hypothesis that tile syn thesis of glycerolipids via the monoacylglycerol pathway may be highly regulated via a variety of lipid second messengers such as phosphatid ic acid and diacylglycerol, as well as by the influx of fatty acids de rive hum high-fat diets, or from the hydrolysis of adipocyte triacylgl ycerol during fasting or diabetes.