Enzyme flavoenzymes

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. ... 

The VAPOR enzymes are flavoenzymes and can be isolated from thermophilic bacilli. They are especially valuable because they allow the regeneration of all four forms of the pyridine nucleotides NADH, NAD+ NAD PH, NADP+ according to the following equations [55,61] ... 

Walpole CSJ, Wrigglesworth R (1987) Oxido-reductases - flavoenzymes. In Page MI, Williams A (eds) Enzyme mechanisms. The Royal Society of Chemistry, London,... 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

The catalytic significance of this observation is not known since no deviation from a two-electron Nemst plot is observed with NADH as reductant and no kinetic studies have been done to compare the rate of the NAD -facilitated comproportionation reaction with the rate of catalytic turnover. No comparable studies on the effect of NADP on the oxidation-reduction potential of ferredoxin-NADP reductase have been, to our knowledge, published. Inasmuch as the physiological role for this enzyme is reduction of the pyridine nucleotide rather than its oxidation, the potential of the enzyme should be significantly lower than that of the pyridine nucleotide couple. Indeed, a value of —445 mV has been determined for this flavoenzyme with the driving force for its reduction being due to a decrease of 90 mV in the one-electron potential of the ferredoxin reductant. This increase... 

In the field of enzymatic oxidations especially, the class of flavoenzymes with bound FAD as cofactor is interesting for synthetic applications. The regeneration of the oxidized FAD within the enzyme can be performed by oxygen. In this case, however, hydrogen peroxide is formed, which drastically diminishes the enzyme stability and activity, rendering it unsuitable for synthesis. 

In the following sections, examples of all the classes of reaction given above will be discussed, with emphasis on those that have been well studied. Many of the enzymes involved in these processes are hemoproteins, but non-heme prosthetic groups are important in oxygenases and are also present in some oxidases. Copper is an important metal in this context, and is present in the oxygen transport protein hemocyanin, and in oxidases such as cytochrome oxidase and laccase. Some flavoenzymes are important too, but will not be covered in this discussion. 

The flavoenzyme D-lactate dehydrogenase from yeast has been reported to contain zinc (134). An apoenzyme can be prepared and reactivated by Zn2+ or Co2+ (135). When yeast is grown in the presence of added Co2+, a Co(II) enzyme is synthesized. The biosynthetic Co(II) enzyme was found to have different catalytic properties compared to the enzyme reactivated from the apoenzyme (136). Only rather fragmentary data have been published on this subject, and the differences in cobalt binding obtained by the two methods of preparation are unknown. 

The enzyme (YerE) involved in the biosynthesis of yersiniose A has been isolated and characterized as a flavoenzyme that catalyzes the attachment of the branched chain to a 3,6-dideoxy-4-hexulose precursor, using pyruvate as the side-chain donor.228 Substrate analogues were prepared as potential inhibitors of the biosynthesis.229... 

To optimize the photoswitchable bioelectrocatalytic features of the protein, site-specific functionalization or mutation of the active site microenvironment is essential. This was accomplished by a semisynthetic approach involving the reconstitution of the flavoenzyme-glucose oxidase with a semisynthetic photoisomerizable FAD cofactor (Scheme 9).1511 The photoisomerizable nitrospiropyran carboxylic acid (24) was covalently coupled to N6-(2-aminoethyl)-FAD (25), to yield the synthetic photoisomerizable nitrospiropyran-FAD cofactor 26a (Scheme 9(A)). The native FAD cofactor was removed from glucose oxidase, and the synthetic photoisomeriz-able-FAD cofactor 26a was reconstituted into the apo-glucose oxidase (apo-GOx), to yield the photoisomerizable enzyme 27a (Scheme 9(B)). This reconstituted protein... 

It is well known that the flavin adenine dinucleotide redox centers of many oxidases are electrically inaccessible due to the insulating effect of the surrounding protein thus, direct electron transfer from the reduced enzyme to a conventional electrode is negligible. In the present work, a variety of polymeric materials have been developed which can facilitate a flow of electrons from the flavin redox centers of oxidases to an electrode. Highly flexible siloxane and ethylene oxide polymers containing covalently attached redox moieties, such as ferrocene, are shown to be capable of rapidly re-oxidizing the reduced flavoenzyme. 

In Table II, kcat/Km values for four naturally occurring flavoenzymes that utilize either NADH or NADPH as substrates are listed. Comparison of these data with those of Table I shows that the oxidation ofN-hexyl-l,4-dihydronicotinamide by flavopapain IV proceeds faster than the reaction illustrated for one of the enzymes, somewhat slower than those illustrated for two of the enzymes, and much slower than that observed for bovine-heart NADH dehydrogenase. The kcat/Km values measured for the reaction of N-hexyl-l,4-dihydronicotinamide as well as those for the N-benzyl-, N-ethyl-, and N-propyl compounds... 

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