Pseudocooperative effects in reactivation of membrane-bound enzymes with phospholipids.

ABSTRACT

The kinetic mechanism for reactivation of membrane-bound enzymes by lipids is analyzed on the basis of multiple equilibriums between the enzyme (which is assumed to contain n identical, non-interacting binding sites) and the lipid. The rate equations derived when only the fully occupied enzyme species ELn (or ELn and the next most highly occupied species ELn-1, ELn-2,..., ELn-i) is catalytically active can fully account for the apparent positive cooperativity observed in the plots of enzyme activity as a function of phospholipid concentration. A general equation for the cases in which more than one lipid species are simultaneously present in the reaction medium is presented which allows to test whether the binding sites are indeed non-interacting, as well as whether the active species have the same catalytic constant (kcat), regardless the nature of the lipid bound to the binding sites. This analysis demonstrates that, in addition to apparent positive cooperativity, more complex curves resembling mixed cooperativity may be obtained with simple systems when interaction between protein subunits (i.e., association-dissociation equilibriums) are present. Finally, some theoretical problems and pitfalls in the interpretation of the experimental results will be discussed.