Activation of reconstituted Escherichia coli outer-membrane phospholipase A by membrane-perturbing peptides results in an increased reactivity towards the affinity label hexadecanesulfonyl fluoride.
The activity of the Escherichia coli outer-membrane phospholipase (OM PLA) is strictly regulated in its natural habitat, the E. coli outer membrane. OM PLA can be reconstituted in phospholipid bilayers, resulting in low specific activity of the enzyme compared to its activity on mixed lipid/detergent micelles. The enzyme can be activated by the addition to these vesicles of the membrane-perturbing peptides polymyxin B, melittin or cardiotoxin resulting in hydrolysis of mainly the sn-1 ester bond of the phospholipids as is also observed in vivo. We used the affinity label hexadecanesulfonyl fluoride to probe the influence of lipid environment on the activity of OM PLA. In detergent and substrate micelles, the rate constant for the sulfonylation of the active-center serine of the purified OM PLA by the affinity label hexadecanesulfonyl fluoride depends on amphiphile concentration. We have reported a similar influence of amphiphile concentration on the activity of the enzyme [Horrevoets, A. J. G. et al. (1989) Biochemistry 28, 1139-1147]. Analysis of the rates of inactivation of OM PLA by hexadecanesulfonyl fluoride in vesicles composed of various phospholipids indicated that activation of the enzyme by membrane-perturbing peptides can be accurately quantified with this affinity label. Our results show that the affinity label hexadecanesulfonyl fluoride can be used to monitor the state of activation of OM PLA in different lipid environments, including non-hydrolyzable substrate analogues. Implications for the in vivo situation are discussed.
Horrevoets
A J
AJ
Department of Enzymology and Protein Engineering, State University of Utrecht, The Netherlands.
Francke
C
C
Verheij
H M
HM
de Haas
G H
GH
D000345:Affinity Labels; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D013450:Sulfones / Q000737:chemistry / Q000494:pharmacology*
D000345:Affinity Labels; D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D013450:Sulfones; C069088:hexadecanesulfonyl fluoride; D010741:Phospholipases A