Davis R C, Wong H, Nikazy J, Wang K, Han Q, Schotz M C
Department of Lipid Research, Veterans Administration Wadsworth Medical Center, Los Angeles, California 90073.
J Biol Chem. 1992 Oct 25;267(30):21499-504.
Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.
利用人脂蛋白脂肪酶（LPL）和大鼠肝脂肪酶（HL）之间的嵌合分子来鉴定导致功能差异的结构元件。基于与胰脂肪酶的紧密序列同源性，LPL和HL都被认为具有双结构域结构，由包含催化性丝氨酸-组氨酸-天冬氨酸三联体的氨基末端（NH2末端）结构域和较小的羧基末端（COOH末端）结构域组成。对含有HL氨基末端结构域和LPL羧基末端结构域的嵌合脂肪酶（HL/LPL）或反向嵌合体（LPL/HL）进行的实验表明，氨基末端结构域决定了这些酶的催化效率（Vmax/Km）。此外，还证明了载脂蛋白C-II对LPL活性的刺激以及1 M NaCl对活性的抑制都源于氨基末端结构域内的结构特征。HL和LPL与血管内皮结合，推测是通过与细胞表面硫酸乙酰肝素蛋白聚糖相互作用。然而，这两种酶在肝素亲和力方面有显著差异。对嵌合脂肪酶的实验表明，肝素结合亲和力主要与羧基末端结构域相关。具体而言，HL和LPL/HL嵌合体都能被0.75 M NaCl从固定化肝素上洗脱，而洗脱LPL和HL/LPL嵌合体则需要1.1 M NaCl。最后，HL在磷脂底物水解方面比LPL更具活性。然而，两种嵌合脂肪酶中磷脂酶与中性脂肪酶活性的比例因异源羧基末端结构域的存在而增加，这表明该结构域强烈影响底物特异性。因此，氨基末端结构域控制这些脂肪酶的动力学参数，而羧基末端结构域调节底物特异性和肝素结合。
