Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.
Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-99.
复制标题
磷脂酶A2工程。
DOI：
10.1021/bi961576x
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发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Sundaralingam,M
中科院分区：
文献类型：
--
作者：
Sekar,K;Yu,BZ;Rogers,J;Lutton,J;Liu,X;Chen,X;Tsai,MD;Jain,MK;Sundaralingam,M
已提出分泌型磷脂酶A2（PLA 2）的天冬氨酸-99对于PLA 2的催化机制和界面活化至关重要。天冬氨酸-99将催化机制（包括催化二联体、推定的催化沃茨W5和W 6以及钙辅因子）连接到氢键网络。后者涉及Y 52、Y 73、结构水和界面活化所需的N-末端区质子。构建了牛胰磷脂酶A2（PLA 2）天冬氨酸+酪氨酸三重突变体（Y 52，73 F/D99 N），测定了其X射线结构，并分析了其动力学特性。对构建的D99 N突变体的动力学特性进行了分析。Y 52，73 F/D99 N突变体的X射线结构表明氢键网络的实质性破坏，包括结构水的损失，类似于先前公开的D99 N突变体的结构中所见[Kumar，A.，Sekharudu，Y. C.的方法，Ramakrishnan，B.，迪普勒角M.的情况，Zhu，H.，蔡美儿D、& Sundaralingam，M.（1994）Protein Sci. 3，2082−2088]。动力学分析表明，这些突变体具有相当大的催化活性，akcat值约为5%，与野生型相比。界面米氏常数的值也几乎没有扰动（约。4-对于D99 N低1倍，对于Y 52，73 F/D99 N略高）。结果表明，氢键网络是不是界面活化至关重要。相反，在突变体中，化学步骤受到了干扰，尽管只是适度的。
The aspartate-99 of secreted phospholipase A2(PLA2) has been proposed to be critical for the catalytic mechanism and interfacial activation of PLA2. Aspartate-99 connects the catalytic machinery (including the catalytic diad, the putative catalytic waters W5 and W6, and the calcium cofactor) to the hydrogen-bonding network. The latter involves Y52, Y73, the structural water, and the N-terminal region putatively required for the interfacial activation. A triple mutant of bovine pancreatic PLA2 with substitutions aspartate plus adjacent tyrosine residues (Y52,73F/D99N) was constructed, its X-ray structure was determined, and kinetic characteristics were analyzed. The kinetic properties of the D99N mutant constructed previously were also further analyzed. The X-ray structure of the Y52,73F/D99N mutant indicated a substantial disruption of the hydrogen-bonding network including the loss of the structural water similar to that seen in the structure of the D99N mutant published previously [Kumar, A., Sekharudu, Y. C., Ramakrishnan, B., Dupureur, C. M., Zhu, H., Tsai, M.-D., & Sundaralingam, M. (1994)Protein Sci. 3, 2082−2088]. Kinetic analysis demonstrated that these mutants possessed considerable catalytic activity with akcatvalue of about 5% compared to WT. The values of the interfacial Michaelis constant were also little perturbed (ca. 4-fold lower for D99N and marginally higher for Y52,73F/D99N). The results taken together suggest that the hydrogen-bonding network is not critically important for interfacial activation. Instead, it is the chemical step that is perturbed, though only modestly, in the mutants.