Phospholipase A2 engineering. The roles of disulfide bonds in structure, conformational stability, and catalytic function.
Phospholipase A2 engineering. The roles of disulfide bonds in structure, conformational stability, and catalytic function.
复制标题
磷脂酶A2工程。
DOI：
10.1021/bi00046a040
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Tsai,MD
中科院分区：
文献类型：
--
作者：
Zhu,H;Dupureur,CM;Zhang,X;Tsai,MD
用定点突变的方法研究了牛胰腺磷脂酶A2(PLA2，在大肠杆菌中过表达)的7个二硫键对该酶的结构、构象稳定性和催化功能的影响。C11-C77、C27-C123、C29-C45、C44-C105、C51-C98、C61-C91和C84-C96这七个二硫键中的每一个通过将两个半胱氨酸(C)残基改变为丙氨酸(A)而被分别删除。用ID和2D质子核磁共振分析了突变体的结构性质，盐酸胍诱导的构象稳定性，以及对DCsPC(1，2-二辛酰甘油-3-磷胆碱)胶束的催化性能。结果得到以下重要发现：(I)除一个突变体(C84A-C96a)外，所有突变体都用与野生型PLA2相同的程序进行了复性和纯化。因此，二硫键通常对PLA2的折叠途径不重要。(Ii)二硫键C11-C77对酶的构象和构象稳定性最重要，因为该二硫键的缺失导致核磁共振性质严重扰动，构象稳定性降低6.2kcal/mol。然而，CHA-C77A突变体的催化功能几乎没有变化，(Iii)除连接钙结合环和螺旋C的二硫键C29-C45外，删除二硫键对PLA2的催化功能影响很小，但C29A-C45A突变体的构象和构象稳定性基本未受影响。(Iv)C29A-C45A突变体的钙结合亲和力降低了10倍或更多。(V)C27-Cl23二硫键的缺失导致酶的构象稳定性意外增加2.4kcal/mol。讨论了PLA2的结构-功能关系和二硫键在蛋白质结构中的作用。二硫键在蛋白质的结构、稳定性和功能中的作用一直是人们非常感兴趣和非常重要的。对这些问题的理解可以导致设计新的蛋白质，或者修改现有的蛋白质。来自牛胰腺的磷脂酶A2(PLA2)1是一种二硫键含量极高的小型酶，它只有123个氨基酸，但与7个二硫键高度交联(该酶不含游离半胱氨酸)。这14种半胱氨酸在40多种氨基酸中高度保守
Site-directed mutagenesis was used to probe the contribution of each of the seven disulfide bonds of bovine pancreatic phospholipase A2 (PLA2, overexpressed in Escherichia coli) to thestructure, conformational stability, and catalytic function of the enzyme. Each of the seven disulfide bonds, C11—C77, C27-C123, C29-C45, C44-C105, C51-C98, C61-C91, and C84-C96, was deleted separately by changing both cysteine (C) residues to alanine (A). The structural properties of the mutants were analyzed by ID and 2Dproton NMR, the conformational stability by guanidine hydrochloride-induced denaturation, and the catalytic property by measuring kinetic parameters toward DCsPC (1, 2-dioctanoylín-glycero-3-phosphocholine) micelles. The results ledto the following significant findings:(i) All but one (C84A—C96A) mutants have been refolded and purified by use of the same procedure for wild-type PLA2. Thus, the disulfide bonds are generally not important to the folding pathway of PLA2.(ii) The disulfide bond C11—C77 is most important to the conformation and conformational stability of the enzyme since deletion of this disulfide bond resulted in greatly perturbed NMR properties and in a decrease of 6.2 kcal/mol in conformational stability. However, the CHA—C77A mutant displayed little change in catalytic function,(iii) The effects of deleting disulfide bonds on the catalytic function of PLA2 are small, except the disulfide bond C29—C45 which connects the calcium binding loop with the helix C. However, the conformation and conformational stability of the C29A—C45A mutant are essentially unperturbed,(iv) The calcium binding affinity of the C29A—C45A mutant was found to decrease by a factor of 10 or greater. This could be the cause for the perturbation in kinetic behavior,(v) Deletion of the C27—Cl23 disulfide bond caused an unexpected increase in the conformational stability of the enzyme by 2.4 kcal/mol. The overall results are discussed in relation to the structure—functionrelationship of PLA2 and the roles of disulfide bonds in protein structures.The roles of disulfide bonds in the structure, stability, and function of proteins have always been of great interest and great importance. Understanding of such problems can lead to designing new proteins, or modifying existingproteins. Phospholipase A2 (PLA2) 1 from bovine pancreas is a small enzyme with unusually high disulfide contents: it consists of only 123 amino acids, but is highly cross-linked with 7 disulfide bonds (the enzyme contains no free cysteines). The 14 cysteines are highly conserved among more than 40