title: On the mechanism of inhibition of methanol dehydrogenase by cyclopropane-derived inhibitors.
abstract: Extraction of cyclopropanol-inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag2O) as a catalyst. Structure elucidation revealed that a C5 3-propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring-closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring-opening. From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring-opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s-1) is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism-based inhibitors. The modified PQQ in cyclopropanone-inactivated MDH is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of MDH with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized-MDH.substrate (MDHox.S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.
authors.affiliation: Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Frank
authors.forename: S H
authors.initials: SH
authors.lastname: van Krimpen
authors.forename: P E
authors.initials: PE
authors.lastname: Verwiel
authors.forename: J A
authors.initials: JA
authors.lastname: Jongejan
authors.forename: A C
authors.initials: AC
authors.lastname: Mulder
authors.forename: J A
authors.initials: JA
authors.lastname: Duine
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D001419:Bacteria / Q000201:enzymology; D003067:Coenzymes / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D003521:Cyclopropanes / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D015394:Molecular Structure; D045542:PQQ Cofactor; D015363:Quinolones / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D003067:Coenzymes; D003521:Cyclopropanes; D015363:Quinolones; D045542:PQQ Cofactor; D003300:Copper; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)