Do membrane-bound enzymes access their substrates from the membrane or aqueous phase: interfacial versus non-interfacial enzymes

For membrane-bound enzymes that act on substrates that partition between th e membrane and aqueous phases, it is possible to imagine two fundamentally different mechanisms. Interfacial enzymes must access their substrate from the membrane phase, in other words substrate in the membrane binds directly to the active site of the enzyme at the membrane without mixing with subst rate molecules in the aqueous phase. On the other hand, non-interfacial enz ymes, either bound to membranes or present in the aqueous phase, must acces s their substrates from the aqueous phase, i.e. substrate in the aqueous ph ase binds directly to the enzyme without mixing with substrates in the memb rane phase. An interfacial mechanism for some enzymes including secreted an d cytosolic phospholipase A(2) and phosphoinositide 3'-hydroxykinase was ri gorously proven by demonstrating that these enzymes processively hydrolyze many phospholipids without desorbing from the surface of vesicles (scooting mode). The non-interfacial mechanism is more difficult to establish becaus e it cannot be addressed by steady-state kinetics. Using a pre-steady-state method in which the enzymatic velocity is measured during the time it take s for substrate to exchange between vesicles? a non-interfacial mechanism w as proven for vesicle-bound plasma platelet activating factor acetylhydrola se. This enzyme prefers more water-soluble phospholipids such as those with sn-2 acetyl or oxidatively truncated fatty acyl chains, and this is readil y explained by the mandatory access of substrate from the aqueous phase. (C ) 2000 Elsevier Science B.V. All rights reserved.