Bezzine S, Ferrato F, Ivanova M G, Lopez V, Verger R, Carrière F
Laboratoire de Lipolyse Enzymatique, CNRS-IFR1, UPR 9025, Marseille, France.
Biochemistry. 1999 Apr 27;38(17):5499-510. doi: 10.1021/bi982601x.
Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.
在一些胰脂肪酶相关蛋白2（PLRP2）中，人胰脂肪酶（HPL）的五个关键氨基酸残基发生了突变，这些蛋白在胆汁盐存在的情况下不能被辅脂酶重新激活。其中一个残基（Y403）参与HPL C末端结构域与辅脂酶之间的直接相互作用。另外四个残基（R256、D257、Y267和K268）参与稳定盖子结构域开放构象的相互作用，盖子结构域也与辅脂酶相互作用。在此，我们制备并表征了三种HPL突变体：HPL Y403N、一种HPL四点突变体（R256G、D257G、Y267F和K268E）以及一种HPL五点突变体（R256G、D257G、Y267F、K268E和Y403N），其中HPL的氨基酸被人PLRP2中存在的氨基酸所取代。辅脂酶重新激活了HPL Y403N突变体和HPL，因此Y403对于脂肪酶 - 辅脂酶相互作用并非必不可少。HPL四点突变体和五点突变体对三辛酸甘油酯均表现出低活性，被胆汁盐（牛磺脱氧胆酸钠，NaTDC）抑制，且不能被辅脂酶重新激活。在4 mM NaTDC存在的情况下，HPL四点突变体与三辛酸甘油酯乳液的界面结合受到抑制，并且添加辅脂酶后也未恢复。蛋白质印迹/蛋白质覆盖免疫测定表明，HPL四点突变体与辅脂酶的相互作用并未消除，因此，HPL四点突变体无法重新激活可能是由于盖子结构域构象阻止了脂肪酶 - 辅脂酶复合物的界面结合。还使用HPL（-盖子）研究了辅脂酶的作用，HPL（-盖子）是一种在盖子结构域内有18个残基缺失的HPL突变体，因此只有一个辅脂酶相互作用位点。HPL（-盖子）对三辛酸甘油酯表现出低活性，被胆汁盐抑制，并且在辅脂酶存在的情况下恢复了其脂肪酶活性。辅脂酶对HPL（-盖子）的重新激活与该突变体与三辛酸甘油酯乳液的强烈界面结合相关。因此，盖子结构域对于HPL的界面结合或脂肪酶 - 辅脂酶相互作用都不是必不可少的。
