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

    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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