Flavine-linked enzymes

P, y-Unsaturated amino acids are not inhibitors of flavin-linked enzymes but are good substrates. These inhibitors appear to be excellent choices as inhibitors of pyridoxal-linked enzyme. A sjmthesis of the parent p, y-unsaturated amino acid, vinyl glycine, is included in this article under the heading Synthesis. ... 

Although propargylamine will irreversibly inhibit both plasma and flavin linked monoamine oxidases, one can still arrange to inhibit one enzyme without affecting the other. Only the flavin-linked enzyme can oxidize tertiary amines, so that acetylenic amines that are tertiary, e.g., pargyline, will inhibit only the flavin-linked enzyme. 

These reactions, which have provided a means of inhibiting the flavin-linked monoamine oxidases, enable us to end on a clinical note. The monoamine oxidases are responsible for the deamination of monoamines such as adrenaline, noradrenaline, dopamine, and serotonin, which act as neurotransmitters. Imbalances in the levels of monoamines cause various psychiatric and neurological disorders Parkinson s disease is associated with lowered levels of dopamine, and low levels of other monoamines are associated with depression. Inhibitors of monoamine oxidases may consequently be used to treat Parkinson s disease and depression. The flavin moiety is covalently bound to the enzyme by the thiol group of a cysteine residue (equation 9.17). The acetylenic suicide inhibitor N,N-dimethyl-propargylamine inactivates monoamine oxidases by alkylating the flavin on N-5.25 A likely mechanism for the reaction is the Michael addition of the N-5 of the reduced flavin to the acetylenic carbon 2... 

Acyl-CoA dehydrogenase is a flavin-linked, membrane-bound enzyme, associated with the mitochondrial respiratory complexes. When FADH2 is produced, it is oxidized by the respiratory chain (Chap. 14). Hydroxyacyl-CoA dehydrogenase is in the mitochondrial matrix, and the NADH produced by the action of this enzyme on hydroxyacyl-CoA compounds contributes to the pool of NADH in the matrix. It is also oxidized by the respiratory chain (Chap. 14). 

Peroxiredoxins Group of antioxidant thioredoxin-dependent enzymes with a catalytic fnnc-tion in the detoxification of cellnlar-toxic peroxides. See Claiborne, A., Ross, R.P., and Parsonage, D., Flavin-linked peroxide rednctases protein-sulfenic acids and the oxidative stress response. Trends Biochem. Sci. 17, 183-186, 1992 Dietz, K-J., Horhing, R, Konig, J., and Baien, M., The function of chloroplast 2-cysteine peroxiredoxin I peroxide detoxification and its regulation, J. Expt. Bot. 53, 1321-1329, 2002 Immenschuh, S. 

The structure of the FMN-linked electron carrier, flavodoxin, has been determined from Clostridium MP by Ludwig and co-workers 59,60) and the corresponding protein from Desuljovibrio vulgaris by Jensen and coworkers (61). Rao and Rossmann 4) showed that, with the coordinates available to them, the standard deviation, o-, between these two structures this structure can be compared with the AMP mononucleotide binding unit of LDH and found that o- = 2.4 A for 39 equivalent atoms. When was 2.1 A for 102 equivalent Cn atoms. They also showed that part of these two quite different proteins were so aligned, it became clear that FMN binds to flavodoxin at a site similar to where AMP binds to LDH. Not only is it unlikely that this is an accidental structural relationship, but furthermore Baltscheffsky 62) has supported a common evolutionary origin for flavin and NAD+-linked enzymes on the basis of a comparison of their redox potentials. 

Volumes XII and XIII, covering Parts B and C of oxidation-reduction enzymes, will include chapters on members of the second great family of dehydrogenases those linked to flavin derivatives. Coverage will also include flavin-linked electron-transferring enzymes. Other sections will cover oxygenases and oxidases, including cytochrome oxidase. Chapters on catalase and peroxidase will complete the volumes. 

For sulfoxides/sulfides, oxidation is catalyzed by cytochrome P-450 and flavin monooxygenases, whereas the reductive metabolism is catalyzed by aldehyde oxidase and/or thiotedocin-linked enzymes. The fiver as well as the gut and bacterial flora are potential sites for the formation of sulfide metabolites. 

Other unsaturated substrate analogs that have been tried as enzyme inhibitors include allyl amine and allyl alcohol. Allylamine is a pseudo-irreversible inhibitor of flavin-linked monoamine oxidase i.e., in the presence of allylamine, the enzyme shows a time-dependent inactivation that cannot be reversed by dialysis. When radiolabeled allylamine is used, radioactivity is incorporated at the same rate as the enzyme is inhibited. However, inhibition is relieved and radioactivity is removed from the enzyme upon incubation with the substrate, benzylamine. 

There are several reagents whose uses may be more restricted than those of inhibitors mentioned above but that nevertheless deserve discussion. Flavin-linked monoamine oxidase has been a fruitful enzyme for the development of new inhibitors. Besides being inhibited by acetylenic amines and olefinic amines, the enzyme is also inactivated by hydra-zides and cyclopropyl amines, e.g., the antidepressant drug tranylcypromine. Both of the latter reagents are first turned over by the enzyme before inhibition ensues, but the mechanisms of inhibition remain obscure. Recently, iV-nitroso compounds have been introduced as irreversible inhibitors of proteolytic enzymes. ... 

These enzymes are highly susceptible to inactivation by y3,y-acetylenic amines. Flavin-linked mitochondrial monoamine oxidase is irreversibly inhibited by the antidepressant drug pargyline. Structure activity studies have shown that the acetylenic unit is crucial and that it has to be /3,y to the nitrogen. ... 

Other flavin-linked oxidase enzymes are also irreversibly inhibited by acetylenes. Lactate dehydrogenase is irreversibly inhibited by 2-hydroxy-3-but3moic acid. As with flavin-linked monoamine oxidase the reaction takes place with the flavin, not with the protein. Finally, flavin-linked... 

Enzymes capable of generating a carbanion or a carbanion-like intermediate adjacent to an acetylene are susceptible to irreversible inhibition by these acetylenic reagents. Flavin-linked and pyridoxal-linked enzymes and isomerases have proved to be good candidates for inhibition by the acetylenic inhibitors. This is the case because these enzymes are generally involved in C—bond abstraction with concomitant carbanion or car-banion-Iike intermediate formation. However, the critical factor is the formation of a free valance adjacent to the acetylenic imit. An enz3rme that could do so is a candidate for inhibition by these inhibitors. Thymi-dylate synthethase, for example, should be irreversibly inhibited by 5-ethynouracil the free valance would be generated adjacent to the acetylene by means of an enzyme-catalyzed nucleophilic addition to the uracil. ... 

Pargyline is a potent irreversible inhibitor of a flavin-linked monoamine oxidase (MAO) and has found clinical application. The latter catalyzes the inactivation of biologically important catecholamines. It forms a covalent bond with the enzyme via the flavin cofactor and the mode of action is believed to be as shown in Scheme 7.1. 

Flavin linked monoamine oxidases are also irreversibly inhibited by molecules that contain an acetylenic moiety. 6 Compounds such as pargyline 1, chlorgyline Z, and deprenyl are irreversible inhibitors of the enzyme and in certain instances they have been used clinically . (Fig. 3)... 

Glycolate which is generated within the chloroplast is exported and further metabolized within the peroxisomes. Here, the flavin mononucleotide (FMN)-linked enzyme glycolate oxidase catalyzes the conversion to... 

Pantothenic acid, sometimes called vitamin B3, is a vitamin that makes up one part of a complex coenzyme called coenzyme A (CoA) (Figure 18.23). Pantothenic acid is also a constituent of acyl carrier proteins. Coenzyme A consists of 3, 5 -adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage. Phosphopantetheine in turn consists of three parts /3-mercaptoethylamine linked to /3-alanine, which makes an amide bond with a branched-chain dihydroxy acid. As was the case for the nicotinamide and flavin coenzymes, the adenine nucleotide moiety of CoA acts as a recognition site, increasing the affinity and specificity of CoA binding to its enzymes. 

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.)...

In the first family, the metal is coordinated by one molecule of the pterin cofactor, while in the second, it is coordinated to two pterin molecules (both in the guanine dinucleotide form, with the two dinucleotides extending from the active site in opposite directions). Some enzymes also contain FejSj clusters (one or more), which do not seem to be directly linked to the Mo centers. The molybdenum hydroxylases invariably possess redox-active sites in addition to the molybdenum center and are found with two basic types of polypeptide architecture. The enzymes metabolizing quinoline-related compounds, and derivatives of nicotinic acid form a separate groups, in which each of the redox active centers are found in separate subunits. Those enzymes possessing flavin subunits are organized as a2jS2A2, with a pair of 2Fe-2S centers in the (3 subunit, the flavin in the (3 subunit, and the molybdenum in the y subunit. 

We next focus on the use of fixed-site cofactors and coenzymes. We note that much of this coenzyme chemistry is now linked to very local two-electron chemistry (H, CH3", CH3CO-, -NH2,0 transfer) in enzymes. Additionally, one-electron changes of coenzymes, quinones, flavins and metal ions especially in membranes are used very much in very fast intermediates of twice the one-electron switches over considerable electron transfer distances. At certain points, the chains of catalysis revert to a two-electron reaction (see Figure 5.2), and the whole complex linkage of diffusion and carriers is part of energy transduction (see also proton transfer and Williams in Further Reading). There is a variety of additional coenzymes which are fixed and which we believe came later in evolution, and there are the very important metal ion cofactors which are separately considered below. 

Riboflavin is the redox component of flavin adenine dinucleotide FAD. It is derived from FAD by hydrolysis of a phosphate ester link. The fully oxidised form of FAD is involved in many dehydrogenaze reactions during which it is converted to the fully reduced form. The fully oxidised state is restored either by another redox enzyme or by interaction with oxygen and hydrogen peroxide is liberated. The one-electron reduced, semiquinone form of FAD, is involved in some electron transfer steps. 

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