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 mi xture at a constant molar ratio of arachidonate/Tween 20 at pH 8.0, th e steady-state velocity on a varied arachidonate concentration did not follow simple Michaelis-Menten-type kinetics and double-reciprocal pl ot analysis gave hyperbolic curves. However, by introducing the concep t of a local pH change, it was possible to analyze the kinetics as sim ple Michaelis-Menten type. The concept of a local pH change implies th at when utilizing an acidic and amphiphilic substance as a substrate, such as arachidonate, the medium around the substrate is acidified wit h 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 phosp holipid model as has been proposed for phospholipase A, [Hendrickson, H. S. and Dennis, E. A. (1984) Kinetic analysis of the dual phospholip id model for phosphalipase A(2), J. Biol. Chem. 258, 5734-5739]. It is concluded that 5-lipoxygenase performs an interfacial reaction in the arachidonatel Tween 20 mixed micelles in the same manner as phospholi pase A(2). The values of K-m were almost constant (about 0.07 molar fr action), even when arachidonate molar ratios were changed in the surfa ce of the mixed micelles. The values for K-s (the association constant of the enzyme to the micelle interface) ranged over 0.21-0.48 mu M. T he V-max was 25.76 mu mol.min(-1).mg(-1). This concept of a local pH c hange could be used extensively with enzymes which utilize both amphip hilic and acidic substances as substrates.