Membrane-bound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase.
Human plasma platelet activating factor acetylhydrolase (pPAF-AH) is a phospholipase A(2) that specifically hydrolyzes the sn-2 ester of platelet activating factor (PAF) and of phospholipids with oxidatively truncated sn-2 fatty acyl chains. pPAF-AH is bound to lipoproteins in vivo, and it binds essentially irreversibly to anionic and zwitterionic phospholipid vesicles in vitro and hydrolyzes PAF and PAF analogues. Substrate hydrolysis also occurs in the absence of vesicles, with a maximum rate reached at the critical micelle concentration. A novel pre-steady-state kinetic analysis with enzyme tightly bound to vesicles and with a substrate that undergoes slow intervesicle exchange establishes that pPAF-AH accesses its substrate from the aqueous phase and thus is not an interfacial enzyme. Such a mechanism readily explains why this enzyme displays dramatic specificity for phospholipids with short sn-2 chains or with medium-length, oxidatively truncated sn-2 chains since a common feature of these lipids is their relatively high water solubility. It also explains why the enzymatic rate drops as the length of the sn-1 chain is increased. pPAF-AH shows broad specificity toward phospholipids with different polar headgroups. Additional results are that PAF undergoes intervesicle exchange on the subminute time scale and it does not undergo transbilayer movement over tens of minutes.
Min
J H
JH
Departments of Biochemistry and Chemistry, University of Washington, Seattle, Washington 98195, USA.
Jain
M K
MK
Wilder
C
C
Paul
L
L
Apitz-Castro
R
R
Aspleaf
D C
DC
Gelb
M H
MH
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D010741:Phospholipases A / Q000097:blood*; D010972:Platelet Activating Factor / Q000378:metabolism; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D008565:Membrane Proteins; D010972:Platelet Activating Factor; D014867:Water; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase