Bezzine S, Roussel A, de Caro J, Gastinel L, de Caro A, Carrière F, Leydier S, Verger R, Cambillau C
Architecture et Fonction des Macromolécules Biologiques, CNRS-IFR1 UPR9039, Marseille, France.
Chem Phys Lipids. 1998 Jun;93(1-2):103-14. doi: 10.1016/s0009-3084(98)00034-6.
Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested: di- and tri-glycerides; phospholipids (PC); etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical PL. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of the three known pancreatic lipase-related protein 1 (PLRP1), whereas Ala and Pro residues are always present at the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic-related protein 1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on tributyrin (1800 U/mg) as well as trioctanoin (2250 U/mg) and its activity is low in the presence of taurodeoxycholate and stimulated in the presence of colipase. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the PLRP1 lipases.
已发现经典犬胰脂肪酶（DPL）和犬胰脂肪酶相关蛋白1（DPLRP1）均由胰腺外分泌腺分泌。这两种蛋白质从犬胰液中纯化至同质，且未观察到DPLRP1对任何测试底物具有显著催化活性：甘油二酯和甘油三酯；磷脂（PC）等。DPLRP1被结晶，其结构通过分子置换解析，并在2.10 Å的分辨率下进行了精修。其结构与在没有任何抑制剂或胶束的情况下测定的经典胰脂肪酶（PL）结构相似。发现控制活性位点入口的盖子结构域呈封闭构象。怀疑DPLRP1中的一个氨基酸取代（丙氨酸178缬氨酸）通过与结合到经典PL的手性C11烷基膦酸酯抑制剂结构中观察到的一个酰基链发生空间冲突而导致酶活性缺失。分别在178和180位存在缬氨酸和丙氨酸残基是三种已知的胰脂肪酶相关蛋白1（PLRP1）的特征，而在PL基因家族的所有其他成员中，丙氨酸和脯氨酸残基总是存在于相同位置。将双突变缬氨酸178丙氨酸和丙氨酸180脯氨酸引入人胰相关蛋白1（HPLRP1）基因，在昆虫细胞中产生了一种表达良好且折叠正确的酶。该酶对三丁酸甘油酯（1800 U/mg）和三辛酸甘油酯（2250 U/mg）具有动力学活性，并且在牛磺脱氧胆酸盐存在下活性较低，在辅脂酶存在下活性受到刺激。因此，我们关于DPLRP1和HPLRP1的发现可能适用于所有PLRP1脂肪酶。
