Dugi K A, Dichek H L, Santamarina-Fojo S
Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Oct 27;270(43):25396-401. doi: 10.1074/jbc.270.43.25396.
Hepatic lipase (HL) and lipoprotein lipase (LPL) are key enzymes that mediate the hydrolysis of triglycerides (TG) and phospholipids (PL) present in circulating plasma lipoproteins. Relative to triacylglycerol hydrolysis, HL displays higher phospholipase activity than LPL. The structural basis for this difference in substrate specificity has not been definitively established. We recently demonstrated that the 22-amino acid loops ("lids") covering the catalytic sites of LPL and HL are critical for the interaction with lipid substrate (Dugi, K.A., Dichek, H.L., Talley, G.D., Brewer, H.B., Jr., and Santamarina-Fojo, S. (1992) J. Biol. Chem. 267, 25086-25091). To determine whether the lipase lid plays a role in conferring the different substrate specificities of HL and LPL, we have generated four chimeric lipases. Characterization of these chimeric enzymes using TG (triolein and tributyrin) or PL (dioleoylphosphatidylcholine (DOPC) vesicles, DOPC proteoliposomes, and DOPC-mixed liposomes) substrates demonstrated marked differences between their relative PL/TG hydrolyzing activities. Chimeric LPL containing the lid of HL had reduced triolein hydrolyzing activity (49% of the wild type), but increased phospholipase activity in DOPC vesicle, DOPC proteoliposome, and DOPC-mixed liposome assay systems (443, 628, and 327% of wild-type LPL, respectively). In contrast, chimeric HL containing the LPL lid was more active against triolein (123% of the wild type) and less active against DOPC (23, 0, and 30%, respectively) than normal HL. Similar results were obtained when the lipase lids were exchanged in chimeric enzymes containing the NH2-terminal end of LPL and the COOH-terminal domain of HL. Exchange of the LPL and HL lids resulted in a reversal of the phospholipase/neutral lipase ratio, establishing the important role of this region in mediating substrate specificity. In summary, the lid covering the catalytic domains in LPL and HL plays a crucial role in determining lipase substrate specificity. The lid of LPL confers preferential triglyceride hydrolysis, whereas the lid of HL augments phospholipase activity. This study provides new insight into the structural basis for the observed in vivo differences in LPL and HL function.
肝脂酶（HL）和脂蛋白脂肪酶（LPL）是介导循环血浆脂蛋白中甘油三酯（TG）和磷脂（PL）水解的关键酶。相对于甘油三酯水解，HL比LPL表现出更高的磷脂酶活性。这种底物特异性差异的结构基础尚未明确确立。我们最近证明，覆盖LPL和HL催化位点的22个氨基酸环（“盖子”）对于与脂质底物的相互作用至关重要（Dugi，K.A.，Dichek，H.L.，Talley，G.D.，Brewer，H.B.，Jr.，和Santamarina-Fojo，S.（1992年）《生物化学杂志》267，25086 - 25091）。为了确定脂肪酶盖子是否在赋予HL和LPL不同底物特异性方面发挥作用，我们构建了四种嵌合脂肪酶。使用TG（三油精和三丁酸甘油酯）或PL（二油酰磷脂酰胆碱（DOPC）囊泡、DOPC蛋白脂质体和DOPC混合脂质体）底物对这些嵌合酶进行表征，结果表明它们相对的PL/TG水解活性存在显著差异。含有HL盖子的嵌合LPL的三油精水解活性降低（为野生型的49%），但在DOPC囊泡、DOPC蛋白脂质体和DOPC混合脂质体测定系统中的磷脂酶活性增加（分别为野生型LPL的443%、628%和327%）。相反，含有LPL盖子的嵌合HL对三油精的活性更高（为野生型的123%），而对DOPC的活性低于正常HL（分别为23%、0%和30%）。当在含有LPL氨基末端和HL羧基末端结构域的嵌合酶中交换脂肪酶盖子时，也获得了类似的结果。LPL和HL盖子的交换导致磷脂酶/中性脂肪酶比率发生逆转，确立了该区域在介导底物特异性方面的重要作用。总之，覆盖LPL和HL催化结构域的盖子在决定脂肪酶底物特异性方面起着关键作用。LPL的盖子赋予优先的甘油三酯水解能力，而HL的盖子增强磷脂酶活性。这项研究为观察到的LPL和HL在体内功能差异的结构基础提供了新的见解。
