Enzyme systems, riboflavin

Flavin Coenzymes, Although many flavin derivatives have been suspected of functioning in oxidizing enzymic systems as prosthetic groups, only two—riboflavin 5-phosphate (flavin mononucleotide, FMN) and flavinade-nine dinucleotide—have been definitely established in enzymic systems. Riboflavin 5 -phosphate (FMN) was identified by Warburg and Christian m ) as a constituent of the old yellow enzyme and its structure elucidated by several workers in different laboratories. Riboflavin, also known as vitamin B2 or lactoflavin, has been synthesized by the following procedure which establishes its structure (147) ... 

Beadle and Tatum had found that irradiation of Neurospora spores produced mutants which were incapable of carrying out certain well-defined chemical reactions, and it was at first supposed that as a result of the destruction of a specific gene, the potentiality for producing a particular enzyme was completely lost. The "wild type" of Neurospora could propagate satisfactorily when biotin was the only vitamin-like substance supplied in the culture medium. Of the many mutant strains produced, however, one needed, in addition to biotin, the vitamin riboflavin. Without a supply of riboflavin in the culture medium this so-called "riboflavinless mutant" would not grow. Since riboflavin is a part of an enzyme system always found in Neurospora, it is an obligatory cell constituent and either has to be produced by the cells themselves (as in the wild type) or supplied exogenously in... 

Because flavin coenzymes are widely distributed in intermediary metabolism, the consequences of deficiency maybe widespread. Because riboflavin coenzymes are involved in the metabohsm of folic acid, pyridoxine, vitamin K, and niacin, deficiency will affect enzyme systems other than those requiring flavin coenzymes. With increasing riboflavin deficiency, tissue concentrations of FMN and FAD fall, as does flavokinase activity, thus further decreasing FMN concentrations. FMN concentrations are decreased proportionally more than FAD concentrations. Decreases in the activities of enzymes requiring FMN generally follow the fall in tissue concentrations, whereas the FAD-dependent enzymes are more variably affected. ... 

No enzyme system which will oxidize kynurenine to hydroxykyiiurenitie has yet been isolated in a cell-free state from any species, and there is some evidence that direct conversion may not normally occur, at least in mammals. Riboflavin was suggested to be concerned in hydroxykynurenine formation at a comparatively early stage (387), and this has been supported by nutritional experiments. 

No active substance could be detected in plants treated with carboxin six weeks after the treatment. The major part of the residue found was sulfoxide. Actually, sulfoxide can occur in the plant in two ways. On the one hand, carboxin is oxidised relatively rapidly in the soil, and the plant takes up its sulfoxide, and, on the other hand, carboxin is metabolised within the plant to sulfoxide, presumably by enzyme systems producing hydrogen peroxide, such as riboflavin or flavin enzymes (Lyr et al., 1975a). It has been proved by extraction with hot dimethyl sulfate that sulfoxide formed in the plant is gradually bound in the form of a water-insoluble complex to lignin and is thus detoxified. No hydrolysis of carboxin in the plant has been observed (Chin et al., 1973). 

FAD is similar in structure to DPN. It differs in that riboflavin replaces the nicotinamide riboside moiety. It is formed when red blood cells are incubated with riboflavin.A purified enzyme system from yeast catalyzes the synthesis of this coenzyme from ATP and flavin mononucleotide. ... 

Because of the slow rate of flavin synthesis by the crude enzyme system, it has been difficult to determine the origin of the four-cari)on unit which condenses with 6,7-dimethyl-8-ribityllumazine to yield the o-xylene ring of the flavin. Two possibilities are apparent, either of which is conristent with the data obtained with radioactive glucose (86) (see Table III and Fig. 4). (a) Acetoin and diacetyl arise from pyruvate in metabolic qrstems and either one will condense chemically with 6,7-dimethyl-8-ribitylluma-zine to yield riboflavin. However, addition of diacetyl to the enzyme system from A. gosaypii did not enhance the incorporation of label from 6,7-... 

It is not known whether the utilization of riboflavin by this organism requires an initial phosphorylation or possibly a cleavage to ribitol. Cells which can attack riboflavin can also oxidize free ribose completely to CO2 and H2O. However, it is concluded that these compounds are oxidized by different enzyme systems since (1) the oxidation of both together is a summation of the oxidation of each separately, and (2) cells not grown on riboflavin and incapable of oxidizing riboflavin still oxidize ribose at a high rate. 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

The water-soluble vitamins generally function as cofactors for metabolism enzymes such as those involved in the production of energy from carbohydrates and fats. Their members consist of vitamin C and vitamin B complex which include thiamine, riboflavin (vitamin B2), nicotinic acid, pyridoxine, pantothenic acid, folic acid, cobalamin (vitamin B12), inositol, and biotin. A number of recent publications have demonstrated that vitamin carriers can transport various types of water-soluble vitamins, but the carrier-mediated systems seem negligible for the membrane transport of fat-soluble vitamins such as vitamin A, D, E, and K. 

Reduced flavins (FADH2, FMNH2, and riboflavin) generated by flavin-dependent reductases have been hypothesized to reduce azo dyes in a nonspecific chemical reaction, and flavin reductases have been revealed to be indeed anaerobic azoreductases. Other reduced enzyme cofactors, for example, NADH, NADH, NADPH, and an NADPH-generating system, have also been reported to reduce azo dyes. Except for enzyme cofactors, different artificial redox mediating compounds, especially such as quinines, are important redox mediators of azo dye anaerobic reduction (Table 1). 

Riboflavin synthase catalyzes the dismutation of 8-D-ribityl-6,7-dimethyllumazine to form the flavin ring system and the general features of the mechanism of this reaction have been known for some time. Recent X-ray structural studies of the enzyme from archaeal organisms such as methanobacteria have shown that the chemical mechanism of action is similar to that of enzymes from eubacteria and eukaryotes although the structures of the enzymes differ greatly <2006JBC1224>. 

The combined dehydrogenation and decarboxylation of pyruvate to the acetyl group of acetyl-CoA (Fig. 16-2) requires the sequential action of three different enzymes and five different coenzymes or prosthetic groups—thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA, sometimes denoted CoA-SH, to emphasize the role of the —SH group), nicotinamide adenine dinucleotide (NAD), and lipoate. Four different vitamins required in human nutrition are vital components of this system thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA). We have already described the roles of FAD and NAD as electron carriers (Chapter 13), and we have encountered TPP as the coenzyme of pyruvate decarboxylase (see Fig. 14-13). 

The final step in riboflavin biosynthesis has been extensively investigated. Incorporation and degradation studies with synthetic (33) using cell-free systems and purified enzymes have shown that two molecules of (33) are utilized to afford one molecule of riboflavin and one molecule of (36). Significantly, the lumazine (33) labelled at the C-6 methyl with deuterium is converted to riboflavin labelled at C-5 and in the C-7 methyl. Based on this and kinetic and spectroscopic data, Plaut has proposed a detailed mechanism for the riboflavin synthetase reaction (B-71MI10402). It is noteworthy that this reaction can also be accomplished non-enzymatically under neutral conditions with the same stereospecificity observed in the enzymic reaction (69CC290). 

Most of the numerous other riboflavin-containing enzymes contain FAD. FAD is an integral part of the biological oxidation-reduction system where it mediates the transfer of hydrogen ions from NAD11 to the oxidized cytochrome system. FAD can also accept hydrogen ions directly from a metabolite and transfer them to either NAD, a metal ion, a heme derivative, or molecular oxygen. The various mechanisms of action of FAD are probably due to differences in protein apoenzymes to which it is bound. 

The central ring of 1-deazaflavins remains a pyrazine in X, a di-hydropyrazine in the two-electron-reduced form, XI, and continues to dominate the chemistry with oxygen. Like the parent riboflavins, and unlike the 5-deazaflavins, the dihydro- 1-deaza system, XI, is reoxidized by 02 in a fraction of a second in air-saturated solutions (Table II) the semiquinone is accessible and 1-deazaFAD enzymes show full catalytic competence with flavoprotein dehydrogenases and oxidases (24). Turnover numbers vary from about 1% to 100% that of cognate FAD-enzymes but this variation reflects the -280 mV vs. —200 mV E° values, respectively, for 1-deazariboflavin vs. riboflavin. The redox steps may or may not limit Vmax with a given enzyme (15, 24). 

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