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J A Jongejan

Publications and source records attributed to J A Jongejan.

31 records · Page 2Linked to original sources
PubMed1989

On the mechanism of inhibition of methanol dehydrogenase by cyclopropane-derived inhibitors.

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.

Alcohol Oxidoreductases↗
PubMed1989

Factors relevant in the reaction of pyrroloquinoline quinone with amino acids. Analytical and mechanistic implications.

In order to reveal the stability of pyrroloquinoline quinone (PQQ) in complex samples, its reaction on incubation with amino acids was followed spectrophotometrically by monitoring oxygen consumption, and with a biological assay. For several alpha-amino acids, the formation of a yellow coloured compound (lambda max = 420 nm) was accompanied by oxygen uptake and disappearance of biological activity from the reaction mixture. The yellow product appeared to be an oxazole of PQQ, the exact structure depending on the amino acid used. Oxazole formation also occurred under anaerobic conditions with concomitant formation of PQQH2, suggesting that PQQ is able to oxidize the presumed oxazoline to the oxazole. Besides the condensation reaction, there is also a catalytic cycle in which an aldimine adduct of PQQ and the amino acid is converted into the aminophenol form of the cofactor and an aldehyde resulting from oxidative decarboxylation of the amino acid. Addition of NH4+ salts, as well as that of certain divalent cations, greatly stimulated both the cyclic and the linear reaction. With basic amino acids, oxazole formation scarcely occurred. However, as oxygen consumption was observed (provided that certain divalent cations were present), conversion of these compounds took place. A reaction scheme is proposed accounting for the products formed and the effects observed. Since NH4+ ions activate several quinoproteins (PQQ-containing enzymes) and divalent cations (Ca2+, Fe2+, and Cu2+) are additional (co)factors in certain metallo quinoproteins, the effects of metal ions observed here could be related to the mechanistic features of these enzymes. Although all oxazoles were converted to PQQ by acid hydrolysis, PQQ was not detected when hydrolysis was carried out in the presence of tryptophan, a compound which appeared to have a deleterious effect on the cofactor under this condition. The results here described explain why analysis methods for free PQQ in complex samples fail in certain cases, or are not quantitative.

Amides↗
PubMed1989

Pyrroloquinoline quinone as cofactor in galactose oxidase (EC 1.1.3.9).

Galactose oxidase from Dactylium dendroides was shown to contain one molecule of covalently bound pyrroloquinoline quinone (PQQ/enzyme molecule. From the spectroscopic characteristics reported for the enzyme forms, a mechanistic role for PQQ could be deduced. In analogy with other quinoproteins, the initial formation of a PQQ-substrate adduct is proposed. Following internal hydrogen transfer, leading to aldehyde product and reduced pyrroloquinoline quinone, reoxidation of the organic cofactor with molecular oxygen could be mediated by the PQQ-liganded copper ion with concomitant formation of hydrogen peroxide. With PQQ as an additional (two-electron) redox center the occurrence of a "superoxidized" enzyme form must be considered. Possible consequences of this view, in relation to a physiological function of the enzyme and interpretation of ESR data, are discussed.

Copper↗
PubMed1989

Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

The three-dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X-ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc-shaped domain. This domain is formed by a circular arrangement of seven topologically identical four-stranded antiparallel beta-sheets, with approximately 7-fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta-sheet scaffold of a central three-stranded antiparallel sheet flanked by two shorter two-stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co-factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co-factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.

Amino Acid Sequence↗
PubMed1988

Dopamine beta-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone.

Treatment of homogeneous dopamine beta-hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper-quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O2. DBH is the first copper-quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed 'hydrazine method' [(1987) FEBS Lett. 221, 299-304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.

Adrenal Medulla↗
PubMed1987

Phenylhydrazine as probe for cofactor identification in amine oxidoreductases. Evidence for PQQ as the cofactor in methylamine dehydrogenase.

Homogeneous methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.3, MADH) from the bacterium Thiobacillus versutus was treated with the inhibitor phenylhydrazine (PH). Derivatization of the cofactor in MADH took place in a fast reaction to give compound I. A different product, compound II, was formed in a slow reaction at high O2 concentrations. The compounds I and II could be removed from the protein by proteolysis with pronase and purified to homogeneity. Products showing identical absorption spectra and chromatographic behaviour were isolated from the reaction mixture after incubating pyrroloquinoline quinone (PQQ) with PH. Upon dissolving in dimethyl sulphoxide, both the enzyme-derived as well as the model-system-derived compounds I and II were nearly quantitatively transformed into PQQ. The conclusion is, therefore, that MADH from T. versutus contains covalently bound PQQ, removable from the protein with pronase, and not a structural analogue of this cofactor without the carboxylic acid groups, as was recently proposed for MADH from Bacterium W3A1 [(1986) Biochem. Biophys. Res. Commun. 141, 562-568]. The properties of compounds I and II suggest that they are the 'azo adduct' and the 'hydrazone adduct' of PH and PQQ at the C(5)-position, respectively. The finding that the reaction of a hydrazine with PQQ can lead to two different products, in enzymes as well as in a model system, has important implications for the interpretation of recent comparative studies aimed at detection of PQQ in amine oxidoreductases with Raman spectroscopy.

Oxidoreductases Acting on CH-NH Group Donors↗
PubMed1986

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase.

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4-dinitrophenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 1H-NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.

Amine Oxidase (Copper-Containing)↗
PubMed1984

Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as prosthetic group.

In addition to the metal ion, copper-containing amine oxidases possess an organic prosthetic group, the nature of which has long been controversial. We show here that in the case of bovine plasma amine oxidase, this second prosthetic group is covalently bound pyrroloquinoline quinone ( PQQ ). Until now the coenzyme PQQ has been found in several bacterial dehydrogenases. Thus the finding reported here is the first example of a quinoprotein oxidoreductase discovered in a eukaryotic organism.

Amine Oxidase (Copper-Containing)↗
PubMed1984

Inactivation of quinoprotein alcohol dehydrogenases with cyclopropane-derived suicide substrates. .

Quinoprotein alcohol dehydrogenases can be inactivated by cyclopropanol, cyclopropanone hydrate, and, depending on whether they can oxidize secondary alcohols, also by cyclopropanone ethyl hemiketal. Only enzyme molecules containing the oxidized coenzyme (PQQ), but not those with the coenzyme in the semiquinone form (PQQH), become inactivated with these compounds. The inactivation process proceeds without proton production or electron acceptor consumption and free radical is not observed in the inactivated enzyme. It could be demonstrated that a stoichiometric relationship exists between enzyme inactivation, PQQ converted, PQQ adduct formed, and cyclopropanol added. Thus the dimeric and monomeric enzyme become fully inactivated with two and one molecule of cyclopropanol, respectively, indicating that the dimeric enzyme contains two independently acting catalytic sites. Inactivation of the enzyme by cyclopropanol and cyclopropanone hydrate produces chromatographically different PQQ adducts. Since cyclopropanemethanol, cyclobutanol and cyclohexanol are not suicide substrates, the inactivation presumably proceeds via a ring opening such as proposed for the metal-ion-catalysed degradation of cyclopropane derivatives. The results are in accordance with our view on the reaction mechanism of these enzymes but not with that of others [Mincey et al. (1981) Biochemistry 20, 7502-7509]. The reasons why their model has to be refuted are discussed.

Alcohol Oxidoreductases↗
PubMed1983

Detection and determination of pyrroloquinoline quinone, the coenzyme of quinoproteins.

A convenient determination of pyrroloquinoline quinone (PQQ) in extracts of purified enzymes is possible with ion-pair chromatography on a HPLC reverse-phase column and with uv detection. However, when culture supernatants have to be analyzed, a fluorescence detection system is more appropriate. Proof for the presence of PQQ can be obtained by treating such a sample with butyraldehyde, which converts the coenzyme into a stable adduct having a suitable retention time in the system. The sensitivity and selectivity of the analysis can be further enhanced by reducing the sample with NaBH4, which produces the dihydrodiol form of the coenzyme (PQQH4) and oxidizing PQQH4 with NaIO4 to a strongly fluorescing compound. A procedure is described for the easy preparation of an apoenzyme from the quinoprotein glucose dehydrogenase of Pseudomonas aeruginosa strains. With this biological test system, very low amounts of PQQ can be detected. However, when PQQ is present in the form of adducts, the analysis has to be performed via reduction to PQQH4, oxidation with NaIO4, and HPLC.

Apoenzymes↗