Interfacial kinetic reaction of human 5-lipoxygenase.
The kinetics of human 5-lipoxygenase were investigated in the presence of Tween 20 using a continuous spectrophotometric assay. Using the mixture at a constant molar ratio of arachidonate/Tween 20 at pH 8.0, the steady-state velocity on a varied arachidonate concentration did not follow simple Michaelis-Menten-type kinetics and double-reciprocal plot analysis gave hyperbolic curves. However, by introducing the concept of a local pH change, it was possible to analyze the kinetics as simple Michaelis-Menten type. The concept of a local pH change implies that when utilizing an acidic and amphiphilic substance as a substrate, such as arachidonate, the medium around the substrate is acidified with an increased concentration of substrate. This concept was explained rationally by two experiments. Consequently, the data were transformed according to a local pH change and analyzed according to a dual phospholipid model as has been proposed for phospholipase A2 [Hendrickson, H. S. and Dennis, E. A. (1984) Kinetic analysis of the dual phospholipid model for phosphalipase A2, J. Biol. Chem. 259, 5734-5739]. It is concluded that 5-lipoxygenase performs an interfacial reaction in the arachidonate/Tween 20 mixed micelles in the same manner as phospholipase A2. The values of Km were almost constant (about 0.07 molar fraction), even when arachidonate molar ratios were changed in the surface of the mixed micelles. The values for Ks (the association constant of the enzyme to the micelle interface) ranged over 0.21-0.48 microM. The Vmax was 25.76 mumol.min-1.mg-1. This concept of a local pH change could be used extensively with enzymes which utilize both amphiphilic and acidic substances as substrates.
Noguchi
M
M
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Miyano
M
M
Kuhara
S
S
Matsumoto
T
T
Noma
M
M
D001094:Arachidonate 5-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010743:Phospholipids; D011136:Polysorbates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D010743:Phospholipids; D011136:Polysorbates; D011994:Recombinant Proteins; D001094:Arachidonate 5-Lipoxygenase