Structural and Functional Effects of Tryptophans Inserted into the Membrane-binding and Substrate-binding Sites of Human Group IIA Phospholipase A2
详细信息 查看全文
- 作者：Kathleen N. Nemec ; Abhay H. Pande ; Shan Qin ; Ramona J. Bieber Urbauer ; Shuhua Tan ; David Moe ; Suren A. Tatulian
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：October 17, 2006
- 年：2006
- 卷：45
- 期：41
- 页码：12448 - 12460
- 全文大小：552K
- 年卷期：v.45,no.41(October 17, 2006)
- ISSN：1520-4995
文摘
Phospholipase A2 (PLA2) enzymes become activated by binding to biological membranes andhydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatorymediators. To understand the functional significance of amino acid residues at key positions, we havestudied the effects of the substitution of Val3 (membrane binding surface) and Phe5 (substrate bindingpocket) of human group IIA PLA2 by tryptophan on the structure and function of the enzyme. Despite theclose proximity of the sites of mutations, the V3W mutation results in substantial enhancement of theenzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structuralanalysis of all three proteins free in buffer and bound to membranes indicates that large differences inactivities result from distinct conformational changes in PLA2s upon membrane binding. Although PLA2and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, theF5W mutant experiences partial distortion of the 
-helical structure presumably resulting from the tendencyof Trp5 to insert into the membrane. Furthermore, whereas the PLA2 and the V3W mutant bind to themembrane at similar and apparently productive-mode orientation, the F5W mutant binds to membraneswith a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutationand the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp3 at the membrane binding face of PLA2 facilitates the proper membranebinding of the enzyme, Trp5 in the internal substrate binding site causes partial unwinding of the N-terminalhelix in order to interact with the membrane.
-helical structure presumably resulting from the tendencyof Trp5 to insert into the membrane. Furthermore, whereas the PLA2 and the V3W mutant bind to themembrane at similar and apparently productive-mode orientation, the F5W mutant binds to membraneswith a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutationand the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp3 at the membrane binding face of PLA2 facilitates the proper membranebinding of the enzyme, Trp5 in the internal substrate binding site causes partial unwinding of the N-terminalhelix in order to interact with the membrane.