Ayvazian L, Crenon I, Hermoso J, Pignol D, Chapus C, Kerfelec B
Unité de Bioénergétique et Ingénierie des Protéines, UPR 9036 CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 9, France.
J Biol Chem. 1998 Dec 11;273(50):33604-9. doi: 10.1074/jbc.273.50.33604.
Among the polar interactions occurring in pancreatic lipase/colipase binding, only one ion pair involving lysine 400 on lipase and glutamic acid 45 on colipase has been described. These residues are strictly conserved among species, suggesting that the ion pair is likely to play an important role. Therefore, in order to prevent this interaction, mutations intended to neutralize or inverse the charge of these residues have been introduced in the cDNAs encoding horse lipase and colipase. The recombinant proteins have been expressed in insect cells, and their catalytic properties have been investigated. In all cases, preventing the formation of the correct ion pair Lys400/Glu45 leads to lipase-colipase complexes of reduced affinity unable to perform an efficient catalysis, notably in the presence of bile salt micelles. Diethyl p-nitrophenyl phosphate inhibition experiments with either mutant lipase or mutant colipase indicate a poor stabilization of the lipase flap. These results suggest that the ion pair plays a critical role in the active conformation of the lipase-colipase-micelle ternary complex by contributing to a correct orientation of colipase relative to lipase resulting in a proper opening of the flap.
在胰脂肪酶/辅脂肪酶结合过程中发生的极性相互作用中，仅描述了一种涉及脂肪酶上的赖氨酸400和辅脂肪酶上的谷氨酸45的离子对。这些残基在物种间严格保守，表明该离子对可能发挥重要作用。因此，为了阻止这种相互作用，已在编码马脂肪酶和辅脂肪酶的cDNA中引入了旨在中和或反转这些残基电荷的突变。重组蛋白已在昆虫细胞中表达，并对其催化特性进行了研究。在所有情况下，阻止正确的离子对Lys400/Glu45形成会导致亲和力降低的脂肪酶-辅脂肪酶复合物，无法进行有效的催化，尤其是在存在胆盐微团的情况下。用突变型脂肪酶或突变型辅脂肪酶进行的对硝基苯磷酸二乙酯抑制实验表明脂肪酶瓣的稳定性较差。这些结果表明，该离子对通过有助于辅脂肪酶相对于脂肪酶的正确定向，从而使瓣适当打开，在脂肪酶-辅脂肪酶-微团三元复合物的活性构象中起关键作用。
