Coleman R A, Wang P, Bhat B G
Department of Nutrition, University of North Carolina, Chapel Hill 27599-7400, USA.
Biochemistry. 1996 Jul 23;35(29):9576-83. doi: 10.1021/bi9602167.
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 respect to MGAT's long-chain acyl-CoA substrate in Triton X-100 mixed micelles. At concentrations between 2.5 and 5.3 mol %, oleic acid stimulated MGAT activity 2-fold, whereas oleic acid inhibited MGAT at concentrations higher than 7.5 mol %. The dependence on palmitoyl-CoA was highly 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 %, Michaelis-Menton kinetics were observed with an apparent k(m) value of about 54 microM for palmitoyl-CoA but with progressively decreasing Vmax values. This effect was not observed with octanoic acid, suggesting that the medium-chain fatty acid is unable to associate stably with the mixed micelle and, thus, cannot substantially alter substrate affinity. When anionic phospholipids were tested, phosphatidic acid, lysophosphatidic acid, phosphatidylserine, and phosphatidylinositol eliminated some of the lag in activation by palmitoyl-CoA. At high molar concentrations of the anionic lipid activators, apparent k(m) values ranged from 77 microM for phosphatidic acid to 196 microM for phosphatidylinositol. Zwitterionic phospholipids had no effect, nor did the non-phospholipid activators bovine serum albumin or sn-1,2-diacylglycerol. CaCl2, but not neomycin or KC1, 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 inhibitor, sphingosine. An altered k(m) for palmitoyl-CoA in the presence of fatty 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 the synthesis of glycerolipids via the monoacylglycerol pathway may be highly regulated via a variety of lipid second messengers such as phosphatidic acid and diacylglycerol, as well as by the influx of fatty acids derived from high-fat diets, or from the hydrolysis of adipocyte triacylglycerol during fasting or diabetes.
为了更好地理解脂肪酸对肝脏单酰甘油酰基转移酶（MGAT）（EC 2.3.1.22）的激活和抑制动力学，我们在Triton X - 100混合胶束中研究了脂肪酸对MGAT的长链酰基辅酶A底物的影响。在2.5至5.3摩尔％的浓度范围内，油酸刺激MGAT活性2倍，而在高于7.5摩尔％的浓度下，油酸抑制MGAT。对棕榈酰辅酶A的依赖性具有高度协同性，希尔常数大于2.4。当油酸浓度低于3摩尔％时，它消除了依赖性曲线中的滞后现象。当油酸浓度高于3摩尔％时，观察到米氏动力学，棕榈酰辅酶A的表观k(m)值约为54 microM，但Vmax值逐渐降低。辛酸未观察到这种效应，表明中链脂肪酸不能与混合胶束稳定结合，因此不能显著改变底物亲和力。当测试阴离子磷脂时，磷脂酸、溶血磷脂酸、磷脂酰丝氨酸和磷脂酰肌醇消除了棕榈酰辅酶A激活中的一些滞后现象。在高摩尔浓度的阴离子脂质激活剂存在下，表观k(m)值范围从磷脂酸的77 microM到磷脂酰肌醇的196 microM。两性离子磷脂没有影响，非磷脂激活剂牛血清白蛋白或sn - 1,2 - 二酰甘油也没有影响。CaCl2可以克服油酸的抑制作用，而新霉素或KCl则不能；因此，脂肪酸的抑制作用似乎不是通过静电相互作用发生的。这些阻滞剂没有改变阴离子磷脂激活剂或抑制剂鞘氨醇所观察到的效果。在脂肪酸或阴离子磷脂存在下，棕榈酰辅酶A的k(m)改变表明长链脂肪酸和磷脂辅因子都可能诱导MGAT的构象变化，从而改变酶对其长链酰基辅酶A底物的亲和力。这些数据进一步支持了这样的假设，即通过单酰甘油途径合成甘油脂质可能通过多种脂质第二信使如磷脂酸和二酰甘油以及来自高脂肪饮食的脂肪酸流入或禁食或糖尿病期间脂肪细胞三酰甘油的水解来高度调节。
