Rajakannan V, Yogavel M, Poi Ming-Jye, Jeyaprakash A Arockia, Jeyakanthan J, Velmurugan D, Tsai Ming-Daw, Sekar K
Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.
J Mol Biol. 2002 Dec 6;324(4):755-62. doi: 10.1016/s0022-2836(02)01132-4.
Phospholipase A(2) catalyses hydrolysis of the ester bond at the C2 position of 3-sn-phosphoglycerides. Here we report the 1.9A resolution crystal structure of the triple mutant K56,120,121M of bovine pancreatic phospholipase A(2). The structure was solved by molecular replacement method using the orthorhombic form of the recombinant phospholipase A(2). The final protein model contains all the 123 amino acid residues, two calcium ions, 125 water molecules and one 2-methyl-2-4-pentanediol molecule. The model has been refined to a crystallographic R-factor of 19.6% (R(free) of 25.9%) for all data between 14.2A and 1.9A. The residues 62-66, which are in a surface loop, are always disordered in the structures of bovine pancreatic phospholipase A(2) and its mutants. It is interesting to note that the residues 62-66 in the present structure is ordered and the conformation varies substantially from those in the previously published structures of this enzyme. An unexpected and interesting observation in the present structure is that, in addition to the functionally important calcium ion in the active site, one more calcium ion is found near the N terminus. Detailed structural analyses suggest that binding of the second calcium ion could be responsible for the conformational change and the ordering of the surface loop. Furthermore, the results suggest a structural reciprocity between the k(cat)(*) allosteric site and surface loop at the i-face, which represents a newly identified structural property of secreted phospholipase A(2).
磷脂酶A(2)催化3-sn-磷脂酰甘油C2位酯键的水解。在此，我们报道了牛胰磷脂酶A(2)的三重突变体K56,120,121M的1.9埃分辨率晶体结构。该结构通过分子置换法，使用重组磷脂酶A(2)的正交晶型得以解析。最终的蛋白质模型包含所有123个氨基酸残基、两个钙离子、125个水分子和一个2-甲基-2,4-戊二醇分子。对于14.2埃至1.9埃之间的所有数据，该模型已精修至晶体学R因子为19.6%（自由R因子为25.9%）。位于表面环中的62 - 66位残基在牛胰磷脂酶A(2)及其突变体的结构中总是无序的。有趣的是，本结构中的62 - 66位残基是有序的，且构象与该酶先前发表的结构有很大不同。在本结构中一个意外且有趣的发现是，除了活性位点中功能重要的钙离子外，在N端附近还发现了另一个钙离子。详细的结构分析表明，第二个钙离子的结合可能是表面环构象变化和有序化的原因。此外，结果表明k(cat)(*)别构位点与i面的表面环之间存在结构互易性，这代表了分泌型磷脂酶A(2)新发现的结构特性。
