Title

Structural effects of covalent inhibition of phospholipase A(2) suggest allosteric coupling between membrane binding and catalytic sites

Authors

Authors

S. A. Tatulian

Abbreviated Journal Title

Biophys. J.

Keywords

BROMO-PHENACYL-BROMIDE; INTERFACIAL ACTIVATION; INFRARED-SPECTROSCOPY; PANCREATIC PHOSPHOLIPASE-A2; DIRECTED MUTAGENESIS; SECONDARY STRUCTURE; GROUP-IIA; MECHANISM; CALCIUM; VENOM; Biophysics

Abstract

Phospholipase A(2) (PLA(2)) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA(2) was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His(48) modification affected PLA(2) structure, its membrane-binding affinity, and the effects of PLA(2) on the membrane structure. The native PLA(2) increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA(2) and the formation of PLA(2)-membrane hydrogen bonding. The inhibited PLA(2) had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible a-helices in the native PLA(2), which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA(2) induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA(2) acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA(2), including the a-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA(2), which contributes to the interfacial activation of the enzyme.

Journal Title

Biophysical Journal

Volume

84

Issue/Number

3

Publication Date

1-1-2003

Document Type

Article

Language

English

First Page

1773

Last Page

1783

WOS Identifier

WOS:000183123000030

ISSN

0006-3495

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