Enzyme Mechanism of Microsomal N,S- Monooxygenase.
Beaty, Narlin Bennet
1980
Abstract
Microsomal N,S-monooxygenase (E.C. 1.14.13.8) is a flavo-protein monooxygenase present in liver microsomes, which uses NADPH and molecular oxygen to hydroxylate a wide variety of amine and sulfur-containing compounds, many of which are drugs and toxic substances. Example amine-containing substrates include atropine, chlorpromazine, desimpramine, methamphetamine, and the potent carcinogen 2-naphthylamine. Sulfur-containing substrates include methimazole, thiobenzamide, mercaptoethanol, and cysteamine. This thesis describes studies of the enzyme including: purification, various physical properties, steady state kinetic analyses, and rapid kinetic studies of the reactions with NADPH, oxygen, and oxygenatable substrates. Also included is a discussion of some kinetic methods of analysis including the pseudo-stationary approximation for the computer solution of stiff differential equations. In this research, I have demonstrated, by stopped-flow kinetic studies, various intermediates in the reaction of microsomal N,S-monooxygenase with its substrates. In so doing, I have clearly established the order of addition of substrates and determined a kinetic mechanism for this enzyme. This mechanism is unique among flavoprotein hydroxylases, in that an air stable flavin-oxygen intermediate (C(4a)-peroxyflavin) is formed prior to the addition of oxygenatable substrate. Therefore, in a reducing environment like that of liver microsomes, the normal state of microsomal N,S-monooxygenase is as a C(4a)-peroxyflavin intermediate, which is considered in other flavin monooxygenases to be a highly reactive intermediate. Thus an explanation for why such a wide variety of compounds are observed to be substrates is that microsomal N,S-monooxygenase already contains the oxygen that will be used for hydroxylation, and any molecule with a suitable active group for hydroxylation, which can come close to the enzyme active site will be oxygenated. This also explains the observation that many substrates, among both the nitrogen and sulfur classes, are oxygenated by the enzyme at a virtually identical rate. The kinetic results which led to the above conclusions are as follows. First, steady state kinetics predict a thermodynamically reversible path between oxygen and NADPH, while NADPH and substrate, and substrate and oxygen are irreversibly connected. Second, substrate does not affect the rate of flavin reduction, which is about 25 times greater than the steady state turnover rate. Third, the formation of a C(4a)-peroxyflavin intermediate from reduced flavoprotein bound to pyridine nucleotide is a kinetically homogeneous process. Furthermore, the oxygen dependence of this reaction leads to the prediction of a Michaelis complex with reduced enzyme and oxygen prior to the formation of the C(4a)-peroxyflavin. The K(,d) for oxygen binding is about 1.0 mM, but is undoubtedly pulled in the forward direction by an irreversible step forming C(4)-peroxyflavin. Fourth, the substrate reaction with C(4a)-peroxyflavin shows a new intermediate with a spectrum like the peroxyflavin, but shifted to slightly lower wavelength. This has been interpreted as a substrate binding spectrum. Fifth, the C(4a)-peroxyflavin has been observed to be stabilized by small concentrations of NADP('+), such that the breakdown of this intermediate to peroxide and oxidized enzyme is negligible.Types
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