Cyclopropylmethyl compounds as probes of the mechanism of hydrogen transfer by NAD(H) and alcohol dehydrogenase

MacInnes, Iain; Nonhebel, D. C.; Orszulik, Stefan T.; Suckling, Colin J.

doi:10.1039/c39820000121

Cited by 24 publications

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“…No evidence of ring-opened products was detected. It was concluded that this establishes that reactions catalyzed by alcohol dehydrogenase do not involve radical intermediates (MacInnes et al, 1982). We do not agree with this argument, although the conclusion in this instance may be correct.…”

Section: Discussionmentioning

confidence: 78%

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Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

Sherry¹,

Abeles²

1985

Biochemistry

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Methanol oxidase isolated from Hansenula polymorpha contains two distinct flavin cofactors in approximately equal amounts. One has been identified as authentic FAD and the other as a modified form of FAD differing only in the ribityl portion of the ribityldiphosphoadenosine side chain. The significance of this finding is as yet unknown. Previous studies have shown that cyclopropanol irreversibly inactivates methanol oxidase [Mincey, T., Tayrien, G., Mildvan, A. S., & Abeles, R. H. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7099-7101]. We have now established that inactivation is accompanied by covalent modification of the flavin cofactor. The stoichiometry of this reaction is 1 mol of cyclopropanol/mol of active flavin. The structure of the covalent adduct was determined by NMR, IR, and UV spectral studies to be an N5,C4a-cyclic 4a,5-dihydroflavin. Reduction of the covalent adduct with NaBH4 at pH 9.0 before removal from the enzyme converted it to the 1-(ribityldiphosphoadenosine)-substituted 4-(3-hydroxypropyl)-2,3-dioxoquinoxaline. Cyclopropyl ring cleavage accompanies inactivation, and covalent bond formation occurs between a methylene carbon of cyclopropanol and N5 of flavin. Methanol oxidase was also reconstituted with 5-deazaflavin adenine dinucleotide (dFAD). Reconstituted enzyme did not catalyze the oxidation of alcohols to the corresponding aldehydes, nor did reduced reconstituted enzyme catalyze the reverse reaction. Incubation of reconstituted enzyme with cyclopropanol resulted in an absorbance decrease at 399 nm, but no irreversible covalent modification of the deazaflavin cofactor. A reversible addition complex between cyclopropanol and dFAD is formed. The structure of that complex was not definitively established, but it is likely that it is formed through the addition of cyclopropoxide to C5 of dFAD. The failure of dFAD-reconstituted methanol oxidase to catalyze the oxidation of substrate, as well as the lack of reaction with cyclopropanol, supports a radical mechanism for alcohol oxidation and cyclopropanol inactivation. Methanol oxidase catalyzes the oxidation of cyclopropylcarbinol to the corresponding aldehyde. No ring-opened products were detected. The failure to form ring-opened products has been used as an argument against radical processes [MacInnes, I., Nonhebel, D. C., Orsculik, S. T., & Suckling, C. J. (1982) J. Chem. Soc., Chem. Commun., 121-122]. We present arguments against this interpretation.

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Section: Discussionmentioning

confidence: 78%

“…Formation of such products would be indicative of the involvement of cyclopropylcarbinol radicals in catalysis (MacInnes et al, 1982). Cyclopropylcarbinol was found to be a substrate for methanol oxidase as measured by rate of oxygen consumption.…”

Section: Reaction Of Methanol Oxidase With Cyclopropylcarbinolmentioning

confidence: 98%

Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

Sherry¹,

Abeles²

1985

Biochemistry

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“…17 . Reactions in the oxidative direction were carried out at 30 "C in O.lM-phosphate buffer, pH 9, and in the reductive direction at 30 "C in O.1M-phosphate buffer, pH 7, following the reaction by the absorption of NADH at 340 nm.…”

Section: Methodsmentioning

confidence: 95%

“…4 6 -4 8 "C at 0.5 Torr) was pure by g.1.c. (1 H, m), 1.4-1.7, 1.7-1.85 (2 H, m), 15 (1 H, s), 2.35-2.6 (2 H, m), 3.3-3.6 (2 H, dd), 5.3-5. 5 (1 H, m), and 5.8-6.0 (1 H, m).…”

Section: Methodsmentioning

confidence: 99%

Latent inhibitors. Part 3. The inhibition of lactate dehydrogenase and alcohol dehydrogenase by cyclopropane-containing compounds

Maclnnes¹,

Nonhebel²,

Orszulik³

et al. 1983

J. Chem. Soc., Perkin Trans. 1

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“…NADH models as electron donorsN AD H model compounds usually transfer hydride (or its equivalent) to substrates with the aid of metal ions or proton acids(MacInnes et a!., 1982).…”

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confidence: 99%

Enzyme Chemistry

1990

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Cyclopropylmethyl compounds as probes of the mechanism of hydrogen transfer by NAD(H) and alcohol dehydrogenase

Abstract: Hydroxyalkylcyclopropanes are oxidised by nicotinamide-dependent horse liver alcohol dehydrogenase without cleavage of the 3-membered ring, implying that radical intermediates are improbable in these reactions.

Cited by 24 publications

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Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

Latent inhibitors. Part 3. The inhibition of lactate dehydrogenase and alcohol dehydrogenase by cyclopropane-containing compounds

Enzyme Chemistry

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