Abstract
Stopped flow kinetic studies have been used to demonstrate three features of the enzymatic mechanism of the microsomal FAD-containing monooxygenase from hog liver. First, in contrast to the bacterial flavin-containing monooxygenases, reduction of the FAD is independent of substrate. Second, the rate of the reaction of reduced enzyme with oxygen to form the C(4a)-peroxyflavin intermediate is independent of substrate. Third, the rate of transformation of the C(4a)-peroxyflavin to oxidized FAD is substrate-dependent. These results are in agreement with the mechanism, determined by steady state kinetic studies (Poulsen, L.L., and Ziegler, D.M. (1979) J. Biol. Chem. 254, 6449-6455), which predicts that the reduced flavin reacts with oxygen before combination with substrate.
Highlights
From the D e ~ ~ r t m oefnB~io~ogicaC~hemistr~, The Lk.it>emityof ~ i c ~ l g fAfnnn, Arbor, Mirhtgan 48109 monooxygenase does notshow such changes (14,15) presumably because the enzyme does bniontd substrate in either the oxidized or the reduced form
Microsomal FAD-containing monooxygenase is a mamma- The purpose of this was to remove a trace contamination by cytolian external flavoprotein monooxygenase (NADPH-requir- chrome b., which otherwise co-purified with microsomal FAD-coning) which catalyzes the monooxygenation of a wide variety of substrates, including secondary and tertiary amines(1, 2 ), hydrazines (31, disubstituted ~-hydroxyamines(4), piperazine-substitutedphenothiazines ( 5 ), thioamides (61, aminothiols (7), thiols (8), and thiocarbamides (8)
Baliou) and consists of the stopped flow spectrophotometer, alogarithmic photometer, a storage oscilloscope, and an X-Y recorder, all interfaced to a Data General addition of substrates based on steady state kinetic results. Their conclusionwas that NADPH and oxygen necessarily preceded the oxygenatable substrate inbinding to theenzyme
Summary
From the D e ~ ~ r t m oefnB~io~ogicaC~hemistr~, The Lk.it>emityof ~ i c ~ l g fAfnnn, Arbor, Mirhtgan 48109 monooxygenase does notshow such changes (14,15) presumably because the enzyme does bniontd substrate in either the oxidized or the reduced form. Dry nitrogen was further purified by passage over BASF catalyst known about the mechanismof related bacterial flavoprotein from Ace Glass Inc. The oxygen-scavengingenzyme protocatechuate monooxygenases (9-13), it is only recently that mechanistic studies have been reported about the mammalianenzyme. P. Baliou) and consists of the stopped flow spectrophotometer, alogarithmic photometer, a storage oscilloscope, and an X-Y recorder, all interfaced to a Data General addition of substrates based on steady state kinetic results.
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