Oxidation of ethylene by cotyledon extracts from Vicia faba L. : Cofactor requirements and kinetics

Improved rates of ethylene oxidation by cell-free preparations from cotyledons of Vicia faba L. have been obtained using cryogenic storage techniques and by developing a method for the hydrolysis of ethylene oxide. Gel permeation chromatography showed that a low-molecular-size fraction was required for activity; accordingly, the kinetics of ethylene oxidation in the presence of this fraction were studied. Reduced pyridine nucleotides could substitute for the low-molecular-size fraction. Activity under a nitrogen atmosphere was 60% lower than in air. The need for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen indicated that the enzyme might be a mixed-function oxidase. Using sufficient NADPH to approach saturation, the apparent Michaelis constant (K m) for ethylene was 1.94±0.38 · 10(-8) M (aqueous phase), and when ethylene was saturating, the K m for NADPH was 3.7 · 10(-5) M. Carbon monoxide was found to inhibit by competing with ethylene, and the inhibitor constant was 5.97 · 10(-7) M in solution. In the presence of excess ethylene and NADPH, activity was highest in phosphate-buffered medium pH 7.9. The bulk of the activity was found in a microsomal fraction.