Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.
The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.
Horrevoets
A J
AJ
Department of Biochemistry, State University of Utrecht, The Netherlands.
Hackeng
T M
TM
Verheij
H M
HM
Dijkman
R
R
de Haas
G H
GH
D002462:Cell Membrane / Q000201:enzymology; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D008823:Micelles; D017830:Octoxynol; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054466:Phospholipases A1; D054467:Phospholipases A2; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity
D003902:Detergents; D008823:Micelles; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; D010740:Phospholipases; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2