Dugi K A, Dichek H L, Talley G D, Brewer H B, Santamarina-Fojo S
Molecular Disease Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
J Biol Chem. 1992 Dec 15;267(35):25086-91.
Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.
脂蛋白脂肪酶（LPL）是启动乳糜微粒和极低密度脂蛋白中甘油三酯水解的关键酶，由多个对正常活性至关重要的功能域组成。LPL的催化结构域介导该酶的酯酶功能，但有人提出不同的脂质结合位点参与LPL在水-脂质界面与乳化脂质底物的相互作用。与胰脂肪酶（PL）一样，LPL含有一个覆盖催化口袋的表面环，该环可能调节底物进入酶活性位点。对该环的二级结构分析揭示了一个螺旋-转角-螺旋基序，其中有两个短的两亲性螺旋，其疏水矩分别为0.64和0.68。为了研究该环在LPL与其底物初始相互作用中的作用，我们利用定点诱变产生了八个构建体，其中该环的两亲性特性发生了改变，并在人胚肾-293细胞中进行了表达。在不改变环预测二级结构的情况下降低两亲性，消除了脂肪酶水解乳化长链脂肪酸甘油三酯（三油精）的能力，但不影响水溶性底物三丁酸甘油酯。用肝脂肪酶的环替换LPL的环，肝脂肪酶的环在22个氨基酸中有15个不同，但也是两亲性的，导致一种保留了三油精和三丁酸甘油酯水解活性的酶的表达。用一个短的四氨基酸肽替换LPL环，该肽可能比22氨基酸环更能直接进入活性位点，使短链脂肪酸甘油三酯的水解增强了两倍多，同时水解乳化底物的能力被消除。因此，LPL环两亲性结构的破坏选择性地降低了乳化脂质底物的水解，而不影响酶的酯酶或催化功能。这些研究表明，带有两个两亲性螺旋的环对于LPL水解长链脂肪酸底物至关重要，为LPL环在脂质-底物相互作用中的作用提供了新的见解。我们提出脂蛋白底物与该环内两亲性螺旋之间的相互作用可能部分决定脂肪酶底物特异性。
