Metabolism of unsaturated fatty acids by RBL-1 5-lipoxygenase: influence of substrate solubility and product inactivation.
Several alternative fatty acid substrates have been employed to characterise the kinetics of rat basophilic leukaemia cell (RBL-1) 5-lipoxygenase. Using arachidonic acid (AA) as substrate, enzymes rates declined at high substrate concentrations (greater than 25 microM) and were associated with pronounced lag phases. The concentrations of AA at which apparent substrate inhibition and lag phases were observed were comparable with those at which AA induced emulsion formation in aqueous media. No evidence for substrate inhibition or lag phases was observed using eicosapentaenoic acid (EPA), a more soluble substrate which did not induce emulsion formation at concentrations up to 100 microM. Reactions catalysed by RBL-1 5-lipoxygenase terminated before exhaustion of substrate. AA and EPA induced time-dependent enzyme inactivation at concentrations 100-fold lower than their apparent Km values for the enzyme. The ability of several fatty acids to induce time-dependent inactivation was directly proportional to their substrate potency. We conclude that apparent substrate inhibition is a consequence of a change from monomeric to micellar substrate which has a lower affinity for the enzyme and that premature termination of the enzyme reactions is a consequence of product-induced enzyme inactivation.
McMillan
R M
RM
Bioscience Department I, ICI Pharmaceuticals, Macclesfield, U.K.
Masters
D J
DJ
Vickers
V C
VC
Dicken
M P
MP
Jacobs
V N
VN
D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001091:Arachidonate Lipoxygenases / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D007700:Kinetics; D016859:Lipoxygenase Inhibitors; D051381:Rats; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014407:Tumor Cells, Cultured
D001095:Arachidonic Acids; D005231:Fatty Acids, Unsaturated; D016859:Lipoxygenase Inhibitors; D016718:Arachidonic Acid; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase