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-1 H-pyrrolo[2,3-,flquinoline-4,5-dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag20) as a catalyst. Structure elucidation revealed that a (25 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-') 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 M D H is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of M D H 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 (MDH,, . 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.Quinoprotein (i.e. PQQ-containing) methanol dehydrogenase (MDH) has been shown to become competitively inhibited by carbonyl-group reagents like cyanide and hydroxylamine [l, 21. The possibility of obtaining a stable oxidized enzyme form in the presence of these inhibitors [I] has been most useful in the detection of an enzyme-substrate complex [3]. More recently, Abeles and coworkers have shown that cyclopropanol acts as an irreversible inhibitor for M D H [4]. Complete inhibition of the monomeric M D H they used was achieved with substoichiometric amounts of cyclopropanol, this being explained by assuming that only the PQQ-semiquinone-containing enzyme molecules are reactive with this compound. However, previous studies conducted in our laboratory have clearly shown that complete inhibition of one dimeric MDH molecule requires two molecules of cyclopropanol while the enzyme should be in its fully oxidized Enzyme. Methanol dehydrogenase (EC 1.1.99.8).form [5]. Since PQQ appeared to become modified in a 1 : 1 stoichiometry and inactivation and spectral changes (above 300nm) occurred...

Shifts in Identity and Activity of Methanotrophs in Arctic Lake Sediments in Response to Temperature Changes

“…In our study, methylotrophs were more dominant in 13 C-labeled DNA libraries from the deep (15-to 20-cm) sediment, accounting for 20.1 to 27.9% of pyrosequencing reads at low temperatures of 10°C and 4°C, than in those from the uppermost (0-to 1-cm) sediment at 21°C. There may be characteristics of deep sediments or their communities that are associated with the interruption of methanotrophic enzymatic reactions, due to the presence of mutations or inhibitors, such as NaCl, EDTA, and HCOONa, or due to gas composition, leading to an excess of methanol that accumulates extracellularly (16,23,24,37). Methylosoma has also been reported to produce formaldehyde that accumulates extracellularly, which might provide 13 C for methylotrophs (48).…”

Functions of Amino Acid Residues in the Active Site ofEscherichia coli Pyrroloquinoline Quinone-Containing Quinoprotein Glucose Dehydrogenase

“…Putative Catalytic Reaction Mechanism of GDH in E. coliTwo types of mechanism have been proposed in the case of MDH for the initial proton-abstraction reaction from methanol: 1) a reaction mechanism via a covalent hemiketal intermediate and 2) a reaction mechanism by a hydride transfer (34,35). At least two lines of evidence supporting the former mechanism have been reported (34,36).…”

Comparison of pathways for biodegradation of monomethyl sulphate in Agrobacterium and Hyphomicrobium species

“…This 2-electron transfer corresponds to oxidation of methanol to formaldehyde but no further, and is consistent with failure of exogenous formaldehyde or formate to elicit a response from the same cells in the electrode. Methanol oxidation by methanol-grown cells was inhibited by cyclopropanol, a known inhibitor of PQQ-dependent methanol dehydrogenase (Dijkstra et al, 1984;Frank et al, 1989;Shimoda & Okura, 1991 (Fig. 5 ) rather than the hydrolytic pathway proposed hitherto (Ghisalba et al, 1985~7, 1986Ghisalba & Kuenzi, 1983;.…”

“…methanol, formaldehyde and formate), the rate of oxygen uptake did not change from the basal rate for either isolate. In both organisms, the MMS-stimulated 0,-uptake was not affected by the presence of cyclopropanol (0.4 mM final concentration), a known inhibitor of PQQ (pyrroloquinoline quinone)-dependent methanol dehydrogenase (Dijkstra et al, 1984;Frank et al, 1989;Shimoda & Okura, 1991). 0,-uptake by methanol-grown Hyphomicrobium sp.…”