Schrag J D, Li Y G, Wu S, Cygler M
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
Nature. 1991 Jun 27;351(6329):761-4. doi: 10.1038/351761a0.
The Ser-His-Asp triad is a well known structural feature of the serine proteases. It has also been directly observed in the catalytic sites of two lipases, whose high-resolution three-dimensional structures have been determined 1,2. Lipases show a wide variety of sizes, substrate and positional specificities, and catalytic rates 3. They achieve maximal catalytic rates at oil-water interfaces. The fungus Geotrichum candidum produces several different forms of lipases, two of which have been purified to homogeneity 4,5. Two lipase genes have been identified, cloned and sequenced 6,7. Both code for proteins of 544 amino acids with a total relative molecular mass of about 60,000 (Mr 60K). The two forms are 86% identical. Their isoelectric points differ slightly, being between 4.3 and 4.6. About 7% of the total Mr is carbohydrate. Until now, only a low resolution structure of GCL has been reported 8, but no high resolution structure has followed. We now report the three-dimensional structure of a lipase from G. candidum (GCL) at 2.2 A resolution. Unlike the other lipases and serine proteases, the catalytic triad of GCL is Ser-His-Glu, with glutamic acid replacing the usual aspartate. Although the sequence similarity with the other two lipases is limited to the region near the active-site serine, there is some similarity in their three-dimensional structures. The GCL is also an alpha/beta protein with a central mixed beta sheet whose topology is similar to that of the N-terminal domain of human pancreatic lipase. As in the other lipases 1,2, the catalytic site is buried under surface loops. Sequence comparisons with proteins from the cholinesterase family suggest that they also contain the Ser-His-Glu triad.
丝氨酸-组氨酸-天冬氨酸三联体是丝氨酸蛋白酶广为人知的结构特征。在两种脂肪酶的催化位点也直接观察到了这一特征，这两种脂肪酶的高分辨率三维结构已被确定[1,2]。脂肪酶呈现出各种各样的大小、底物特异性、位置特异性和催化速率[3]。它们在油水界面达到最大催化速率。白地霉能产生几种不同形式的脂肪酶，其中两种已被纯化至同质[4,5]。已鉴定、克隆并测序了两个脂肪酶基因[6,7]。这两个基因编码的蛋白质均含544个氨基酸，总相对分子质量约为60,000（Mr 60K）。这两种形式的蛋白质有86%的序列相同。它们的等电点略有不同，在4.3至4.6之间。总Mr的约7%为碳水化合物。到目前为止，仅报道了白地霉脂肪酶（GCL）的低分辨率结构[8]，但尚未有高分辨率结构的报道。我们现在报道了白地霉脂肪酶（GCL）在2.2 Å分辨率下的三维结构。与其他脂肪酶和丝氨酸蛋白酶不同，GCL的催化三联体是丝氨酸-组氨酸-谷氨酸，谷氨酸取代了通常的天冬氨酸。尽管与其他两种脂肪酶的序列相似性仅限于活性位点丝氨酸附近的区域，但它们的三维结构存在一些相似之处。GCL也是一种α/β蛋白，有一个中央混合β折叠，其拓扑结构与人胰脂肪酶的N端结构域相似。与其他脂肪酶一样[1,2]，催化位点埋藏在表面环之下。与胆碱酯酶家族蛋白质的序列比较表明，它们也含有丝氨酸-组氨酸-谷氨酸三联体。
