HUMAN HEPATIC AND LIPOPROTEIN-LIPASE - THE LOOP COVERING THE CATALYTIC SITE MEDIATES LIPASE SUBSTRATE-SPECIFICITY
HUMAN HEPATIC AND LIPOPROTEIN-LIPASE - THE LOOP COVERING THE CATALYTIC SITE MEDIATES LIPASE SUBSTRATE-SPECIFICITY
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DOI：
10.1074/jbc.270.43.25396
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发表时间：
1995-10-27
影响因子：
4.8
通讯作者：
SANTAMARINAFOJO, S
中科院分区：
文献类型：
--
作者：
DUGI, KA;DICHEK, HL;SANTAMARINAFOJO, S
肝脂酶(HL)和脂蛋白脂酶(LPL)是调节循环血浆脂蛋白中甘油三酯(TG)和磷脂(PL)水解的关键酶。相对于三酰甘油的水解，HL具有比LPL更高的磷脂酶活性。底物专一性的这种差异的结构基础尚未确定。我们最近证明了覆盖LPL和HL催化位置的22个氨基酸环对于与脂质底物(Dugi，K.A.Dichek，H.L.，Tley，G.D.，Breyer，H.B.，Jr.和Santamarina-Fojo，S.(1992)J.Biol)的相互作用是关键的。化学。267、25086-25091)。为了确定脂肪酶盖是否在赋予HL和LPL不同底物特异性方面发挥作用，我们产生了四种嵌合脂肪酶。用TG(三酸甘油酯和三丁酸甘油酯)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了表征，结果表明它们的相对PL/TG水解性显著不同。在DOPC囊泡、DOPC蛋白脂质体和DOPC-混合脂质体体系中，含有HL封端的嵌合LPL降低了三油酸脂的水解活性(野生型的49%)，但提高了磷脂酶的活性(分别为野生型的443、628和327%)。相反，与正常HL相比，含有LPL盖子的嵌合BL对三油酸甘油酯的活性更高(野生型的123%)，对DOPC的活性更低(分别为23%、0和30%)。当脂肪酶盖在含有LPL的NH2末端和HL的COOH末端的嵌合酶中交换时，也得到了类似的结果。LPL和HL LID的交换导致磷脂酶/中性脂肪酶比率的逆转，确立了该区域在调节底物特异性方面的重要作用。LPL的LID提供优先的甘油三酯水解，而HL的LID增强磷脂酶活性。这项研究为体内观察到的LPL和HL功能差异的结构基础提供了新的见解。
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 BL 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.