Flavin/imine mechanism

Figure 5.68 Flavin/imine mechanism of UDP galactopyranose mutase. The direction of curvature of bound FADH as shown in the X-ray crystal structure is sketched.

There are demethylases which act like amine oxidases that are dependent in their mechanism on their cosubstrate flavine adenine dinucleotide (FAD). So far, lysine-specific demethylase 1 (LSDl) is the only representative of this class [62]. LSDl, as an amine oxidase leads to oxidation of the methylated lysine residue, generating an imine intermediate, while the protein-bound cosubstrate FAD is reduced to FAD H2. In a second step, the imine intermediate is hydrolyzed to produce the demethylated histone lysine residue and formaldehyde. Importantly the reduced cosubstrate is regenerated to its oxidized form by molecular oxygen, producing hydrogen peroxide (Figure 5.7) [62, 63]. 

Over the years there have been a number of mechanistic proposals for substrate oxidation by TMADH. An early proposal considered a carbanion mechanism in which an active site base deprotonates a substrate methyl group to form a substrate carbanion [69] reduction of the flavin was then achieved by the formation of a carbanion-flavin N5 adduct, with subsequent formation of the product imine and dihydroflavin. A number of active site residues were identified as potential bases in such a reaction mechanism. Directed mutagenesis and stopped-flow kinetic studies, however, have been used to systematically eliminate the participation of these residues in a carbanion-type mechanism [76-79], thus indicating that a proton abstraction mechanism initiated by an active site residue does not occur in TMADH. Early proposals also invoked the trimethylammonium cation as the reactive species in the enzyme-substrate complex, owing to the high (9.81) of free... 

Three mechanisms have been proposed for the reactivation of N-CBA-inactivated MAO these are illustrated in Scheme 19 (102). in pathway (A), the aldehyde group of the active site adduct forms a Schiff base with the activator amine, which is followed by base-catalyzed /3-eIimination of the imine of the covalent adduct and subsequent nonenzymic hydrolysis. The product has been identified as acrolein (103). Air oxidation of the unblocked reduced flavin cofactor then leads to active enzyme. The base may be an amino acid residue or a second molecule of the activator. Pathway (B) is similar to the first, without prior formation of the Schiff base. An S 2 displacement reaction is suggested by path-... 

Crystal structures rationalize the kinetic mechanisms of DAAOs. They show that RgDAAO does not contain the lid that covers the active site of pkDAAO and is proposed to regulate product dissociation. In RgDAAO, the side chain of Tyr238 might play the same role. Stopped-flow studies have shown flavin reduction without intermediates and, in cases where the imine product dissociates slowly enough, an imine—reduced enzyme charge-transfer complex." " ... 

Figure 7.19 The mechanism of N-oxidation relies on a flavin moiety of MFO undergoing reduction by NADPH.The intermediate formed reacts with molecular oxygen to form a hydroperoxide. The terminal oxygen of a hydroperoxide is electrophilic and can react with nucleophiles such as the lone pair of an imine leading to imine oxide formation. The flavin unit undergoes a ring-opening and ring-closure sequence and finally eliminates water to give back the oxidized flavin unit that can continue in the catalytic cycle. Based on Figure 13 in reference [3].

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