Simons J W, van Kampen M D, Ubarretxena-Belandia I, Cox R C, Alves dos Santos C M, Egmond M R, Verheij H M
Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, The Netherlands.
Biochemistry. 1999 Jan 5;38(1):2-10. doi: 10.1021/bi981869l.
In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.
在本研究中，我们已确定来自猪葡萄球菌的脂肪酶中存在钙的高亲和力结合位点。通过等温滴定量热法，我们表明该酶每分子酶结合一个钙，解离常数为55微摩尔。与存在钙离子时的活性相比，脱辅基酶的残余活性在10℃时为65%，在40℃时几乎为零。基于与其他葡萄球菌脂肪酶以及来自嗜热链杆菌和谷壳假单胞菌的脂肪酶的一级序列比对，并结合定点诱变，天冬氨酸354和357可被鉴定为钙配体。对D357E变体的动力学测量表明，用谷氨酸替代天冬氨酸357会使对钙离子的亲和力降低30倍。在位置357处引入赖氨酸、天冬酰胺或丙氨酸以及在位置354处引入赖氨酸或天冬酰胺会产生不依赖钙的变体。等温滴定量热法证实了钙结合的丧失。尽管D357K、D357N和D357A变体不结合钙，但在室温下，它们在有钙存在时的活性与野生型脂肪酶几乎相同，但在升高温度时，这些不依赖钙的脂肪酶活性降低。在整个温度范围内，D354K和D354N变体在有钙存在时的活性明显低于野生型酶，且与野生型脱辅基酶的活性相当。我们的结果表明，钙的结合对葡萄球菌脂肪酶（可能还有其他脂肪酶）的结构稳定很重要，并且基于与另一种脂肪酶有限的结构同源性来构建不依赖钙的变体是可行的。
