Adduct, substrate-flavin

Fig. 18. Covalent substrate-coenzyme adducts in flavin biochemistry...

Flavin oxidases include d- and l-amino acid oxidases, and some amine oxidases, although others are quinoproteins (Section 9.8.3). In these enzymes, the flavin is reduced by dehydrogenation of the substrate, byway of an intermediate substrate-flavin adduct, as occurs in the dehydrogenases (Section 7.3.3). 

MCAD is one of the best studied flavoprotein dehydrogenases (10). In this enzyme, the pro-R a-hydrogen of the acyl-CoA thioester is removed by the catalytic base Glu376 and the pro-R 3-hydrogen of the substrate is transferred directly to flavin N5 as a hydride (11) (Fig. 2a). MCAD is inactivated by a range of acyl-CoA derivatives. One such compound is methylenecyclopropylacetyl-CoA which acts as a suicide inhibitor by forming a covalent adduct with flavin N5 (see Further Reading for more information). 

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. 

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... 

The three-dimensional structure of the complex of D-amino acid oxidase with the substrate analog benzoate has been determined. The carboxyl group of the inhibitor is bound by an arginine side chain (Fig. 15-11) that probably also holds the amino acid substrate. There is no basic group nearby in the enzyme that could serve to remove the a-H atom in Eq. 15-26 but the position is appropriate for a direct transfer of the hydrogen to the flavin as a hydride ion as in Eq. 15-23.161/162/257 In spite of all arguments to the contrary the hydride ion mechanism could be correct However, an adduct mechanism is still possible. 

In the active site of a hydroxylase, an OH group can be transferred from the peroxide to a suitable substrate (Eq. 18-42). Although radical mechanisms are likely to be involved, such hydroxylation reactions can also be viewed as transfer of OH+ to the substrate together with protonation on the inner oxygen atom of the original peroxide to give a 4a - OH adduct. The latter is a covalent hydrate which can be converted to the oxidized flavin by elimination of H20. This hydrate is believed to be the third spectral intermediate identified during the action of p-hydroxybenzoate hydroxylase 286 287 290... 

PQQ and the other quinone prosthetic groups described here all function in reactions that would be possible for pyridine nucleotide or flavin coenzymes. All of them, like the flavins, can exist in oxidized, half-reduced semiquinone and fully reduced dihydro forms. The questions to be asked are the same as we asked for flavins. How do the substrates react How is the reduced cofactor reoxidized In nonenzymatic reactions alcohols, amines, and enolate anions all add at C-5 of PQQ to give adducts such as that shown for methanol in Eq. 15-51, step a 444,449,449a Although many additional reactions are possible, this addition is a reasonable first step in the mechanism shown in Eq. 15-51. An enzymatic base could remove a proton as is indicated in step b to give PQQH2. The pathway for reoxidation (step c) might involve a cytochrome b, cytochrome c, or bound ubiquinone.445 446... 

Disaccharides and even some insoluble polysaccharides are substrates, but not monosaccharides. Cellobiose oxidase is unusual among flavoproteins, as it stabilises the red anionic flavin semiquinone and forms a sulphite adduct, yet appears to produce the superoxide anion as its primary reduced oxygen product. 

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. 

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) ... 

The structures of EX and El were deduced by resolution of El into apoenzyme and free flavin-substrate adduct. The structure of this adduct was determined as 5-cyanoethy 1-1,5-dihydro FAD and that of EX was deduced to be a cationic imine resulting from elimination of NO2" from the initial 5-nitroethy 1-1,5-dihydro FAD adduct formed in the process controlled by k2 by nucleophilic attack of nitroethane carbanion on the position of oxidized flavin. The chemistry of flavin reduction by nitroethane carbanion at the active site of D-amino acid oxidase is given by the following scheme (Equation 19) in which the kinetically important... 

The isoalloxazine moiety of the flavin cofactor forms the catalytic heart of a flavoenzyme. It can undergo one- and two-electron redox transitions and form covalent adducts with substrates and protein residues. The redox properties of the flavin cofactor are modulated by the protein environment. In free flavin, the one-electron reduced state is thermodynamically unstable. Elavoenzymes, however, can stabilize the neutral or anionic semiquinone state (Fig. Ic) (44), and they can pass the electrons one at a time to other redox centers. 

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... 

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