Franken P A, Van den Berg L, Huang J, Gunyuzlu P, Lugtigheid R B, Verheij H M, De Haas G H
Department of Enzymology and Protein Engineering, University of Utrecht, The Netherlands.
Eur J Biochem. 1992 Jan 15;203(1-2):89-98. doi: 10.1111/j.1432-1033.1992.tb19832.x.
Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.
猪胰磷脂酶A2（PLA2）中的两个甲硫氨酸残基都已被亮氨酸取代，但其仍保留全部酶活性。无甲硫氨酸突变体在大肠杆菌中作为Cro-LacZ融合蛋白表达，通过用溴化氰进行化学裂解从中释放出前体PLA2。通过将人血小板磷脂酶A2（HP-PLA2）中的单个Met8替换为亮氨酸残基，并在HP-PLA2序列之前的位置引入一个甲硫氨酸，证明了溴化氰对缺乏甲硫氨酸残基的蛋白质进行裂解具有普遍适用性。该蛋白在大肠杆菌中作为68 kDa的Cro-LacZ融合蛋白表达。溴化氰裂解释放出HP-PLA2片段，该片段在体外进行了再氧化。[Met8----Leu]HP-PLA2在水溶液中为单体，酶活性需要毫摩尔范围内的钙离子，在pH 8左右具有最佳活性。对溴苯甲酰溴能迅速使该酶失活，钙离子具有保护作用。分别在1,2-二辛酰-sn-甘油-3-磷酸甘油的纯胶束以及牛磺脱氧胆酸盐和1,2-二辛酰-sn-甘油-3-磷酸甘油的混合胶束中发现了最高比活性，分别为2400 U/mg和9300 U/mg。在混合胶束中，对二油酰磷脂的活性按磷脂酰甘油＞磷脂酰乙醇胺＞＞磷脂酰胆碱的顺序降低。该酶作为底物对单体1,2-二庚酰-sn-甘油-3-磷酸胆碱活性较低，但对胶束活性较高，在临界胶束浓度时活性有明显跃升。用底物类似物正十六烷基磷酸胆碱的胶束溶液测定了HP-PLA2、猪胰PLA2和黑眉眼镜蛇毒PLA2与脂质/水界面的结合。与胰PLA2（0.2 mM）和蛇毒PLA2（0.03 mM）相比，HP-PLA2具有较高的表观解离常数（2 mM）。在牛磺脱氧胆酸盐和1,2-十二酰-sn-甘油-3-磷酸胆碱的混合胶束中，测试了2-十四烷酰氨基己醇-1-磷酸胆碱及其磷酸甘油和磷酸乙醇胺衍生物的R和S对映体对HP-PLA2的竞争性抑制作用。S对映体只是弱抑制剂，而R对映体是强效抑制剂。抑制能力取决于极性头部基团的性质，按磷酸胆碱＜＜磷酸乙醇胺＜磷酸甘油的顺序增加。最佳抑制剂（R）-2-十四烷酰氨基己醇-1-磷酸甘油与底物相比，与HP-PLA2活性位点的结合力强2200倍。
