Loomes K M
Biochemistry and Molecular Biology Group, School of Biological Sciences, University of Auckland, New Zealand.
Eur J Biochem. 1995 Jun 1;230(2):607-13. doi: 10.1111/j.1432-1033.1995.tb20602.x.
Bile-salt-activated lipase belongs to the cholinesterase alpha/beta-hydrolase-fold family of proteins. Here, we have investigated the structural organisation of the human isoform by mapping tryptic cleavage sites using limited proteolysis and by expression studies using a recombinant truncated variant. Two accessible regions in the tertiary structure were identified. The first is defined by a tryptic cleavage at Lys429 and lies within the alpha/beta-hydrolase fold in bile-salt-activated lipase between a central beta-sheet and an active-site histidine residue, as deduced from sequence similarity across the cholinesterases and known structural properties. This region exhibits a proteolytic and topological similarity to the lid region in pancreatic lipase. The other accessible region in the tertiary structure is defined by a tryptic cleavage at Arg520 and occurs within a catalytically non-essential segment Leu519-Gln535, as identified by expression of a truncated variant which lacks the C-terminus starting from Leu519. This region is consistent with an interdomain region between the cholinesterase-related part of the protein structure and the unique proline-rich C-terminal repeats. Both protease-sensitive regions appear to occur at domain borders, and, therefore, are consistent with a multi-domain structure. The truncated variant was fully functional as a lipase and as a bile-salt-stimulated esterase. However, compared to the full-length enzyme, the truncated variant showed an increased susceptibility to limited proteolysis, suggesting that the C-terminal repeats may regulate proteolytic degradation of the protein.
胆汁盐激活脂肪酶属于胆碱酯酶α/β水解酶折叠家族蛋白。在此，我们通过有限蛋白酶解定位胰蛋白酶切割位点以及使用重组截短变体进行表达研究，对人同工型的结构组织进行了研究。确定了三级结构中的两个可及区域。第一个区域由赖氨酸429处的胰蛋白酶切割所界定，位于胆汁盐激活脂肪酶的α/β水解酶折叠内，介于中央β折叠片层和活性位点组氨酸残基之间，这是根据胆碱酯酶之间的序列相似性和已知结构特性推断出来的。该区域与胰脂肪酶中的盖子区域表现出蛋白水解和拓扑相似性。三级结构中的另一个可及区域由精氨酸520处的胰蛋白酶切割所界定，位于催化非必需片段亮氨酸519 - 谷氨酰胺535内，这是通过表达从亮氨酸519开始缺失C末端的截短变体确定的。该区域与蛋白质结构中胆碱酯酶相关部分和独特的富含脯氨酸的C末端重复序列之间的结构域间区域一致。两个蛋白酶敏感区域似乎都出现在结构域边界处，因此与多结构域结构一致。截短变体作为脂肪酶和胆汁盐刺激的酯酶具有完全功能。然而，与全长酶相比，截短变体对有限蛋白酶解的敏感性增加，这表明C末端重复序列可能调节该蛋白的蛋白水解降解。
