Title

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A(2)

Authors

Authors

K. N. Nemec; A. H. Pande; S. Qin; R. J. B. Urbauer; S. H. Tan; D. Moe;S. A. Tatulian

Comments

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Abbreviated Journal Title

Biochemistry

Keywords

INTERFACIAL ACTIVATION; INFRARED-SPECTROSCOPY; SNAKE-VENOM; ALPHA-HELIX; PHOSPHATIDYLCHOLINE VESICLES; ANTIBACTERIAL PROPERTIES; SECONDARY; STRUCTURE; X-RAY; PROTEINS; ENZYME; Biochemistry & Molecular Biology

Abstract

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.

Journal Title

Biochemistry

Volume

45

Issue/Number

41

Publication Date

1-1-2006

Document Type

Article

Language

English

First Page

12448

Last Page

12460

WOS Identifier

WOS:000241107000006

ISSN

0006-2960

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