Substrate flavin adduct formation

Both Equations 12 and 13 involve adduct formation between substrate and flavin, and both require removal of carbon-bound hydrogen as a proton. They differ substantially, however, in that adduct formation in Equation 12 occurs through the substrate carbon atom, whereas in equation 13 adduct formation occurs through nucleophilic attack of — XH on the flavin nucleus (50). Equation 14 involves no intermediate and can be visualized as a hydride transfer reaction. 

Studies of nitroalkane oxidation by n-amino acid oxidase (55) and glucose oxidase 49, 56) have provided strong evidence both for intermediate substrate carbanions and for subsequent covalent adduct formation between these and the N position of the flavin nucleus. The rationale for using nitroalkanes can be seen in the following reaction stoichiometries for D-amino acid oxidase (55) ... 

Experimental support for the mechanism of Eq. 15-26 has been obtained using D-chloroalanine as a substrate for D-amino acid oxidase.252-254 Chloro-pyruvate is the expected product, but under anaerobic conditions pyruvate was formed. Kinetic data obtained with a-2H and a-3H substrates suggested a common intermediate for formation of both pyruvate and chloro-pyruvate. This intermediate could be an anion formed by loss of H+ either from alanine or from a C-4a adduct. The anion could eliminate chloride ion as indicated by the dashed arrows in the following structure. This would lead to formation of pyruvate without reduction of the flavin. Alternatively, the electrons from the carbanion could flow into the flavin (green arrows), reducing it as in Eq. 15-26. A similar mechanism has been suggested for other flavoenzymes 249/255 Objections to the carbanion mechanism are the expected... 

Having shown that general-base-catalyzed carbanion formation precedes the oxidation step in flavin oxidation of the second class of carbon acids, the question arises as to how the electron pair moves from the carbanion to flavin. Covalent addition of carbanion to Flox followed by a base-catalyzed elimination reaction is one possibility (13). Addition to the 4a- (Equation 17) and 5-position (Equation 18) would appear to be feasible and, a priori, it would seem reasonable to expect that these adducts could undergo an elimination to yield oxidized substrate and reduced flavin. Nucleophilic addition of SO J to... 

Reactions were studied under the pseudo first-order condition of [substrate] much greater than [initial dihydroflavin]. Under these conditions, the reactions are characterized by a burst in the production of Flox followed by a much slower rate of Flox formation until completion of reaction. The initial burst is provided by the competition between parallel pseudo first-order Reactions a and b of Scheme 3. These convert dihydroflavin and carbonyl compound to an equilibrium mixture of carbinolamine and imine (Reaction a), and to Flox and alcohol (Reaction b), respectively. The slower production of Flox, following the initial burst, occurs by the conversion of carbinolamine back to reduced flavin and substrate and, more importantly, by the disproportionation of product Flox with carbinolamine (Reaction c followed by d). Reactions c and d constitute an autocatalysis by oxidized flavin of the conversion of carbinolamine back to starting dihydroflavin and substrate. In the course of these studies, the contribution of acid-base catalysis to the reactions of Scheme 3 were determined. The significant feature to be pointed out here is that carbinolamine does not undergo an elimination reaction to yield Flox and lactic acid (Equation 25). The carbinolamine (N(5)-covalent adduct) is formed in a... 

Equation 26. Electron-deficient flavins will also oxidize nitroalkane anions in model reactions (12). The observation (11) that nitromethane anion and FloXEt yield a stable 4a-adduct is evidence that 4a-adducts are not on the reaction path for nitroalkane oxidation. That the blocking of the N(5)-position of flavin (i.e., FloxEt) prevents oxidation of nitromethane would, however, be in accord with the requirement for an N(5)-adduct (11). The nitroalkane reaction with flavoenzyme has been used to implicate N(5)-adducts as intermediates in the oxidation mechanism of amino acid oxidases. However, it must be understood that nitroalkane anions differ significantly from the carbanions generated from a normal substrate. The nitroalkane anion on loss of its pair of electrons would provide an impossibly unstable carbonium ion, whereas in the case of the amino acid anion an internal electron release obviates carbonium ion formation. 

The initial step of the two-electron-transferring reactions is the removal of a proton from the substrate, followed by the intermediate formation of an adduct between the substrate and prosthetic group at N-5 of the flavin. This undergoes cleavage to yield dUiydroflavin and the oxidized product, which is commonly a carbon-carbon double bond. The reduced flavin is then reoxidized by reaction with an electron-transferring flavoprotein, as discussed above, or in some cases by reaction with nicotinamide nucleotide coenzymes. 

One-electron transfer from the substrate amino group to flavin (FI) results in the formation of the aminium radical and the flavin radical anion (FC) (Scheme 15). Deprotonation of the aminium radical to yield an a-aminoalkyl radical followed by a second electron transfer to the flavin radical anion will result in the formation of the reduced flavin and iminium ion. Alternatively the iminium ion can be formed by path d in Scheme 15 this involves formation of a covalent adduct which can... 

By considering the above-mentioned solution studies and the refined three-dimensional structure of the S. cerevisiae flavocytochrome 62 active site, Lederer and Mathews proposed a scheme for the reverse reaction (the reduction of pyruvate) (39). They did not discuss how the transfer of electrons took place except to say that the structure did not rule out the possibility of a covalent intermediate (39). Ghisla and Massey (116) considered the anionic flavin N5 to be too close to the pyruvate carbonyl (3.7 A) without the formation of a covalent adduct taking place. Covalent intermediates between substrate and flavin have been observed for lactate oxidase (117, 118) and o-amino acid... 

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... 

One class of mechanism-based MAO inhibitors includes the unsaturated alkylamines (propargylamine analogs) (Table II). Although the kinetics of enzyme inactivation for these compounds are consistent with a mechanism-based inhibitor, in only a few cases has the chemical mechanism and site of protein modification been determined. Pargyline (iV-benzyl-N-methyl-2-propynylamine) is a classic example. Pargyline reacts stoichiometrically and irreversibly with the MAO of bovine kidney, with protection from inactivation afforded by substrate benzylamine (91). Furthermore, the reaction involves bleaching of the FAD cofactor at 455 nm and the formation of a new absorbing species at 410 nm and a covalent adduct of inactivator with flavin cofactor (92). 

The mechanism of UGM is of great interest and is still not completely resolved. Both a single-electron transfer mechanism and a nucleophilic mechanism have been proposed (Scheme 28). Both mechanisms involve the formation of a substrate—flavin N5 adduct, which has been trapped during turnover. This could arise due to direct nucleophilic attack of the reduced flavin on the sugar substrate. Alternatively, this adduct could arise from one-electron transfer from the reduced flavin to an oxocarbenium ion generated by elimination of UDP, followed by radical recombination of the flavin semiquinone and hexose radical. " An oxocarbenium ion is a proposed intermediate based on positional isotope exchange experiments and studies with substrate analogues, possible. 

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