Acetylenes, oxidation inactivation

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

A contrasting mode of flavoprotein reactivity with an acetylenic inactivator occurs in the reaction of 2-hydroxy-3-butynoate (13, Fig. 15) with a number of a-hydroxy acid oxidizing enzymes. This process is exemplified by the inactivation of L-lactate oxidase from Mycobacterium smegmatis, an enzyme which catalyzes the oxidative decarboxylation of lactate to yield acetate, carbon dioxide, and water (Walsh, 1979, p. 408). Incubation of 13 with lactate oxidase leads to inactivation of the enzyme with a partition ratio that varies from 110 in the... 

Agents that are oxidatively activated and inactivate the enzyme by covalently binding to it include (a) diverse sulfur compounds (e.g., carbon disulfide , parathion -, diethyldithiocarbamate , isothiocyanates , thioureas , thiophenes , tie-nilic acid - - , and mercaptosteroids , (b) halo-genated structures such as chloramphenicoF- , A-monosubstituted dichloroacetamides , and N-(2-p-nitrophenethyl)dichloroacetamide , (c) alkyl and aryl olefins and acetylenes such as... 

Figure 7.8. The oxidation of terminal acetylenes, and even some internal acetylenes, results in the formation of ketene intermediates that react with water to give the carboxylic acids (see Chapter 6). It appears that the ketenes also react with active-site residues, inactivating the P450 enzyme that forms them. The hydrogen that undergoes a 1,2-migration during the oxidation reaction is indicated by a star. The structures of 2-ethynylnaphthalene and 10-undecynoic acid, both of which inactivate P450 enzymes, at least in part by this mechanism, are shown.

P450-catalyzed oxidation of terminal acetylenes to substituted acetic acids (Chapter 6) is more prone to result in heme alkylation than the oxidation of terminal olefins. The structure-activity relationships for the acetylene reaction are similar to those for terminal olefins, except that there are fewer instances in which the reaction does not result in errzyme inactivation. For example, P450 is inactivated by phenylacetylene but not delectably by styrene ", and P450 is inactivated by internal acetylenes, albeit without heme adduct formation, but not by internal olefins 22 , Catalytic oxidation of the acetylenic function is required for enzyme inactivation and terminal acetylenes give heme adducts analogous to those obtained with terminal olefins - 259 jhe salient difference in the adducts obtained with acetylenes and olefins... 

Strategies to convert selective P450 substrates to suicide inactivators by the incorporation of a suitable activatable function at the position oxidized are not always successful. For instance, the introduction of an acetylenic moiety into the chemical template Af-(3,5-dichloro-4-pyridyl)-... 

Williams, D.E., A.S. Muerhoff, N.O. Reich, C.A. CaJacob, P.R. Ortiz de Montellano, and B.S.S. Masters (1989). Prostaglandin and fatty acid o) and (co-l) oxidation in rabbit lung. Acetylenic fatty acid mechanism based inactivators as specific inhibitors. J. Biol. Chem. 264, 749-756. 

The oxidation of terminal acetylenes, like that of monosubstituted olefins, often results in inactivation of the P450 enzyme involved in the oxidation. In some instances, this inactivation involves reaction of the ketene metabolite with nucleophilic residues on the protein [196, 197], but in other instances it involves alkylation of the prosthetic heme group (Fig. 4.31). Again, as found for heme alkylation in the oxidation of olefins, the terminal carbon of the acetylene binds to a pyrrole nitrogen of the heme and a hydroxyl is attached to the internal carbon of the triple bond. Of course, as one of the two m-bonds of the acetylene remains in the adduct, keto-enol equilibration yields a final adduct structure with a carbonyl on the original internal carbon of the triple bond [182, 198]. It is to be noted that the oxidation of terminal triple bonds that produces ketene metabohtes requires addition of the ferryl oxygen to the imsubstituted, terminal carbon, whereas the oxidation that results in heme alkylation requires its addition to the internal carbon. As a rale, the ratios of metabolite formation to heme alkylation are much smaller for terminal acetylenes than for olefins. 

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