Open AccessInterfacial kinetic reaction of human 5‐lipoxygenase
Author(s) -
NOGUCHI Masato,
MIYANO Masashi,
KUHARA Satoru,
MATSUMOTO Takashi,
NOMA Masana
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18867.x
Subject(s) - chemistry , micelle , phospholipase a2 , phospholipid , substrate (aquarium) , lipoxygenase , michaelis–menten kinetics , kinetics , reaction rate constant , molar concentration , phospholipase a , chromatography , critical micelle concentration , steady state (chemistry) , analytical chemistry (journal) , enzyme , organic chemistry , enzyme assay , biochemistry , aqueous solution , membrane , oceanography , physics , quantum mechanics , geology
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 A 2 [Hendrickson, H. S. and Dennis, E. A. (1984) Kinetic analysis of the dual phospholipid model for phosphalipase A 2 , 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 A 2 . The values of K m 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 K s (the association constant of the enzyme to the micelle interface) ranged over 0.21–0.48 μM. The V max was 25.76 μmol · 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.