Inactivation of Escherichia coli outer-membrane phospholipase A by the affinity label hexadecanesulfonyl fluoride. Evidence for an active-site serine.
The Escherichia coli outer-membrane phospholipase A (OM PLA) is a membrane-bound acyl hydrolase with a broad substrate specificity. In order to obtain more insight into the mechanism of action of this enzyme, we designed an active-site-directed inhibitor for OM PLA on the basis of the known substrate specificity as a first step in the elucidation of the catalytic mechanism of this enzyme. The inhibitor, hexadecanesulfonyl fluoride, consists of a long hydrocarbon chain for high-affinity binding by the enzyme and a sulfonyl fluoride moiety as a reactive group. The kinetics of the inactivation of OM PLA by hexadecanesulfonyl fluoride were studied in Triton X-100 micelles. Inactivation is very fast, specific and shows the same characteristics with respect to acyl specificity, pH profile and metal ion requirement as the activity of OM PLA on substrates. Incubation of OM PLA with a stoichiometric amount of hexadecanesulfonyl fluoride leads to a total and irreversible loss of enzyme activity, resulting from the sulfonylation of Ser144. This Ser144, which we suggest to be the active-site serine of OM PLA, is part of the sequence HDSNG, whereas in the water-soluble serine proteases and lipases the structural motif GXSXG is normally encountered. On the basis of the kinetics of inactivation of OM PLA by hexadecanesulfonyl fluoride, we discuss a possible catalytic mechanism of the enzyme.
Horrevoets
A J
AJ
Department of Enzymology, State University of Utrecht, The Netherlands.
Verheij
H M
HM
de Haas
G H
GH
D000345:Affinity Labels; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017830:Octoxynol; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D011092:Polyethylene Glycols; D013450:Sulfones / Q000737:chemistry / Q000494:pharmacology*; D013501:Surface-Active Agents
D000345:Affinity Labels; D001425:Bacterial Outer Membrane Proteins; D013450:Sulfones; D013501:Surface-Active Agents; D011092:Polyethylene Glycols; C069088:hexadecanesulfonyl fluoride; D017830:Octoxynol; D010741:Phospholipases A; D003488:Cyanogen Bromide