Flavin-Substrate Complexes

After some remarks on flavoquinone the structural discussion of possible reaction intermediates will start with the radical (= complex ofFlox + e ) and continue with dihydroflavin (= complex ofFlox + H"), flavin-substrate complexes (Flox + R") and flavin-oxygen complexes (Flox + OOH ). Scheme 6 illustrates the a-input and -output positions for formation and decay of these intermediates. 

Another problem encountered in flavin-substrate complexes is the determination of the flavin redox state, as outlined above. It is, of course, the spectral behavior, which decides the assignment. If the complex is of a-type, its spectral behavior will simulate a dihydroflavin. Determination of protonation state is then the problem to be resolved. If on the other hand it is a 7c-complex, two possibilities are open according to Sequence A. In the case of nicotinamide substrates, both types of 7u-complexes, the red Flox-NiCredH and the green NiCox-Flred, can be verified in chemical as well as enzymic systems (7). But it remains to be seen whether they are indispensable prerequisites for exchange of redox equivalents between nicotinamides and flavins. Maximal 7c-overlap is apparently favourable for oxidoreduction, but it is an open question whether any other steric conditions might do as well. Scheme 10 accounts for what may be called a-transfer of 2e -equivalents within a tc-complex. 

The enzymes of this type that have been characterized contain some type of redox-active cofactor, such as a flavin (3), or a metal ion (heme iron, non-heme iron, or copper), or both (4-6). Our understanding of the mechanism of these enzymes is most advanced in the case of the heme-containing enzyme cytochrome P450. But in spite of the availability of a crystal structure of an enzyme-substrate complex (7) and extensive information about related reactions of low molecular weight synthetic analogues of cytochrome P450 (8), a detailed picture of the molecular events that are referred to as "dioxygen activation" continues to elude us. 

The endoplasmic reticulum electron-transport system (NADPH-cytochrome P-450 reductase) can also generate [18]. This system, which is often responsible for the metabolism of foreign compounds, is selectively distributed in a wide variety of cell types. Its presence in hepatocytes is particularly important, since drugs are often metabolised at this site. In this system, a single electron is transferred from reduced flavin to a cytochrome P-450-substrate complex. A second electron is then transferred through this complex to O2. Production of O - may occur through auto-oxidation of the partially reduced flavin cofactor or because of uncoupling of electrons from the enzyme-substrate complex to 02 [19]. 

Fig. 10. Proposed mechanism for halosubstrate oxidation (route 1) or elimination (route 2) via a carbanion intermediate. The formation of the enzyme-substrate complex is followed by abstraction of the hydrogen at C-2 by the active site base. The carbanion intermediate can then undergo oxidation to form halopyruvate via route 1, or can eliminate halide to form pyruvate via route 2. E, Enzyme S, substrate FI, flavin B, active site base.

Over the years there have been a number of mechanistic proposals for substrate oxidation by TMADH. An early proposal considered a carbanion mechanism in which an active site base deprotonates a substrate methyl group to form a substrate carbanion [69] reduction of the flavin was then achieved by the formation of a carbanion-flavin N5 adduct, with subsequent formation of the product imine and dihydroflavin. A number of active site residues were identified as potential bases in such a reaction mechanism. Directed mutagenesis and stopped-flow kinetic studies, however, have been used to systematically eliminate the participation of these residues in a carbanion-type mechanism [76-79], thus indicating that a proton abstraction mechanism initiated by an active site residue does not occur in TMADH. Early proposals also invoked the trimethylammonium cation as the reactive species in the enzyme-substrate complex, owing to the high (9.81) of free... 

Substrate bond breakage by wild-type TMADH is too fast to be followed using the stopped-flow method in the regime where both His-172 and trimethylamine in the enzyme-substrate complex are deprotonated (that is pH 10). Consequently, our tunneling studies have focused on the compromised mutant enzymes H172Q and Y169F ( 10-fold and 40-fold reduction in the limiting rate constant for flavin reduction compared with wild-type enzyme, respectively). 

The reduced enzyme-substrate complex reacts with molecular oxygen forming a flavin hydroperoxide, which hydroxylates 2-aminobenzyl-CoA at the 5-position at 80 s (Scheme 33). In the absence of the reductase flavin, an aromatic product is obtained. However, in its presence, the hydroxylated intermediate is reduced rapidly and undergoes tautomerization to give the product 2-amino-5-oxocyclohex-l-enecarboxyl-CoA. 

When 5-enolpyruvylshikimate-3-phosphate was added to an anaerobic sample of reduced FMN and chorismate synthase, the substrate was immediately converted to chorismate, followed by slow formation of a flavin radical. Similarly, when (6R)-6-fluoro-5-enolpyruvylshikimate-3-phosphate was added to an identical sample, rapid formation of the radical was observed. It was suggested that the reduced flavin/enzyme complex is not stable in the presence of substrate or substrate analogue and is oxidized by an unidentified species to yield the flavin radical. 

A different type of phenomenon is observed for the two specific Rapid signals which are obtained from xanthine (Fig. 3). Whilst the no complex detected types of signal are still seen with very low xanthine concentrations (88), these are replaced at higher xanthine concentration by a different signal (76, 88, see also Section V D and Fig. 4) which seems to represent a mixture of variable amounts of two complexes of reduced enzyme (76, 78). These complexes are, apparently, not with a product derived from the xanthine molecule which originally reduced molybdenum (76). Instead they involve a further xanthine molecule, this remaining un-oxidized in the complex. Ultimately, when molybdenum has been reoxidized (via iron and flavin), this substrate molecule having... 

We have not so far mentioned the Phase III increase in the Rapid signal (Fig. 5). It seems (67) that Phase II represents over reduction of molybdenum to Mo(IV), possibly by substrate radicals (see Section V H). The system then comes towards thermodynamic equilibrium by interaction between reduced active enzyme molecules and oxidized inactive ones (67, cf. 64). As Mo(IV) of the former is oxidized to Mo(V), during Phase III, so iron or flavin of the inactive enzyme is reduced. Later, in Phase IV, molybdenum of the inactive enzyme is reduced also to give the Slow signed. Alloxanthine, which as noted above, forms a stable complex with Mo(IV), seems to abolish both the slow phase in the 450 nm bleaching of the enzyme by xanthine and the Phase III increase in Rapid signal (91). 

Finally, in the case of inhibitory substrate analogues such as allo-xanthine, strong evidence has recently been presented that these bind to molybdenum in reduced xanthine oxidase (33). If the enzyme is reduced with xanthine, then treated anaerobically with alloxanthine and finally exposed to air, catalytic activity is lost. Though flavin and iron in the final product are in the oxidized state, there are significant spectral differences between it and the native enzyme. These are believed (33) due to reduction of molybdenum from Mo(VI) to Mo(IV) and complexing of... 

Upon purification of the XDH from C. purinolyticum, a separate Se-labeled peak appeared eluting from a DEAE sepharose column. This second peak also appeared to contain a flavin based on UV-visible spectrum. This peak did not use xanthine as a substrate for the reduction of artificial electron acceptors (2,6 dichlor-oindophenol, DCIP), and based on this altered specificity this fraction was further studied. Subsequent purification and analysis showed the enzyme complex consisted of four subunits, and contained molybdenum, iron selenium, and FAD. The most unique property of this enzyme lies in its substrate specificity. Purine, hypoxanthine (6-OH purine), and 2-OH purine were all found to serve as reductants in the presence of DCIP, yet xanthine was not a substrate at any concentration tested. The enzyme was named purine hydroxylase to differentiate it from similar enzymes that use xanthine as a substrate. To date, this is the only enzyme in the molybdenum hydroxylase family (including aldehyde oxidoreductases) that does not hydroxylate the 8-position of the purine ring. This unique substrate specificity, coupled with the studies of Andreesen on purine fermentation pathways, suggests that xanthine is the key intermediate that is broken down in a selenium-dependent purine fermentation pathway. ... 

The addition of cofactors to antibodies is a sure means to confer a catalytic activity to them insofar as this cofactor is responsible for the activity. Indeed for many enzymes, the interaction with cofactors such as thiamins, flavins, pyridoxal phosphate, and ions or metal complexes is absolutely essential for the catalysis. It is thus a question there of building a new biocatalyst with two partners the cofactor responsible for the catalytic activity, and the antibody which binds not only the cofactor but also the substrate that it positions in a specific way one with respect to the other, and can possibly take part in the catalysis thanks to some of its amino acids. 

LSDl, also known as BHCllO, is the first lysine specific demethylase that was discovered. It has been assigned to group I of lysine demethylases (KDMl) [90, 91]. LSDl contains an amine oxidase domain responsible of the enzymatic activity and has been isolated as a stable component from several histone modifying complexes. The enzymatic characterization of this protein revealed that FAD (flavine adenine dinucleotide) is required as a cofactor for the removal of the methyl group. Furthermore, LSDl requires a protonated nitrogen in order to initiate demethylation so that this enzyme is only able to demethylate mono- or dimethylated substrates but not trimethylated substrates [98, 99]. 

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