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 the 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 substrate molecules in the aqueous phase. On the other hand, non-interfacial enzymes, either bound to membranes or present in the aqueous phase, must access their substrates from the aqueous phase, i.e. substrate in the aqueous phase binds directly to the enzyme without mixing with substrates in the membrane phase. An interfacial mechanism for some enzymes including secreted and cytosolic phospholipase A(2) and phosphoinositide 3'-hydroxykinase was rigorously 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 because 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 takes for substrate to exchange between vesicles, a non-interfacial mechanism was proven for vesicle-bound plasma platelet activating factor acetylhydrolase. This enzyme prefers more water-soluble phospholipids such as those with sn-2 acetyl or oxidatively truncated fatty acyl chains, and this is readily explained by the mandatory access of substrate from the aqueous phase.
Gelb
M H
MH
Department of Chemistry, University of Washington, Seattle 98195, USA. gelb@chem.washington.edu
Min
J H
JH
Jain
M K
MK
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; C067460:1-oleoyl-2-(12-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D009327:4-Chloro-7-nitrobenzofurazan