Flavin residue

Oxygenases and dehydrogenases (Table 1) contain flavin residues, whose isoalloxazin ring system is reversibly reduced by 2e to 1,5-dihydroflavin. Glucose oxidase (MW = 186,000) contains two isoalloxazin systems and moreover two buried disulflde bridges. The d.p. reduction peak is 0.1 V more positive than the peak of free FAD, due to the protein moiety. Cholesterol oxidase shows similar behavior with nearly the same C/1/2, -0.32 vs. SCE (pH 7), of a reversible step which was studied extensively at the d.m.e. and the h.m.d.e. 

Schabort and Potgieter (1971) showed that each of the J -cyclopiazonate oxidocyclases contain one covalently linked molecule of flavin per molecule of enzyme. The purified enzymes had a yellow color, and their ultraviolet and visible spectra showed maxima at 276, 366, and 450 nm. The addition of sodium dithionite or of jSCA caused the disappearance of the 450 nm peak because of the reduction of the flavin moiety. The flavin residues released from the protein molecules by proteolytic digestion with pronase showed absorption peaks at 262, 370, and 450 nm. The released flavin residues, separated by paper chromatography, were found to contain a covalently bound amino acid or small peptide. It was concluded that in the native enzyme, the flavin was bound by covalent linkage to the protein and that, during digestion with pronase, proteolysis stops at points determined by the specificity of the pronase or by structural or steric hindrance by the flavin. Pentose determination on the flavin residues showed that the flavins were dinucleosides. 

All the complexes consist of several subunits (Table 2) complex I has a flavin mononucleotide (FMN) prosthetic group and complex II a flavin adenine dinucleotide (FAD) prosthetic group. Complexes I, II, and III contain iron-sulphur (FeS) centers. These centers contain either two, three, or four Fe atoms linked to the sulphydryl groups of peptide cysteine residues and they also contain acid-labile sulphur atoms. Each center can accept or donate reversibly a single electron. 

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD nicotinamide adenine dinucleotide FAD, flavin adenine dinucleotide TDP, thiamin diphosphate.)...

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

In contrast to the flavin-dependent monoamine oxidases, SSAO/VAP-1 has evolved to hydroxylate a tyrosine residue in the active site which is further oxidized to the quinone state by oxygen in the presence of copper ion releasing hydrogen peroxide [28-30]. The primary amine in the substrate (R-NH2, Scheme 1) forms a Schiff-base with the quinone carbonyl group, which through a series of steps ultimately releases the aldehyde product. 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

The first and most extensively examined system was the hydrolytic enzyme papain. A variety of isomeric a-bromoacetylisoalloxazines were used to selectively tether a flavin moiety to the active site cysteine residue. Different isomeric linkages were proposed to allow orientations of the flavin relative to the substrate binding site which would favor reactions with a bound substrate [65]. 

Carell has recently presented the study of a flavin amino acid chimera to model riboflavin in DNA photolyases [68]. This amino acid LI (Fig. 20) was synthesized in an enantiopure fashion by building the alloxazine ring onto the epsilon amine of lysine. This coenzyme chimera was applied to the problem of repairing DNA damage caused by UV irradiation. LI was incorporated into an 21-residue peptide, P-1, possessing the sequence of the DNA-binding domain of the helix-loop-helix transcription factor MyoD. 

In contrast to the use of self-assembly reactions and metal ion coordination preferences to direct the construction of mixed cofactor systems, the use of SPPS or selective chemical ligation allows for the direct covalent attachment of cofactors for the construction of mixed cofactor systems within de novo design. Figure 11 shows the flavocy-tochrome maquette constructed by Dutton and co-workers (149) using a flavin moiety covalently attached to a unique cysteine residue inside a four helix bundle with bis-histidine binding sites for heme... 

Phosphoric acid molecules can form acid-anhydride bonds with each other. It is therefore possible for two nucleotides to be linked via the phosphate residues. This gives rise to dinucleotides with a phosphoric acid-anhydride structure. This group includes the coenzymes NAD(P) " and CoA, as well as the flavin derivative FAD (1 see p. 104). 

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]. 

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