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Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2.

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_name

Biochemistry

journal_title

Biochemistry

authors

Nemec KN,Pande AH,Qin S,Bieber Urbauer RJ,Tan S,Moe D,Tatulian SA

doi

10.1021/bi061440r

subject

Has Abstract

pub_date

2006-10-17 00:00:00

pages

12448-60

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2.