Homogeneous methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.3, MADH) from the bacterium Thiobaciflus 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 02 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 incubatng 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 W3Al [(1986) Biothem. Biophys. Res. Commun. 141, 562-5681. 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.
Application of the so-called hexanol extraction procedure for PQQ determination, originally based on detachment of the cofactor from quinoproteins and conversion into PQQ-S,S-dihexyl ketal, leads in several cases to a number of products due to uncontrollable esterification. The present modified procedure, detaching the covalently bound cofactor and converting it into 4-hydroxy-5-hexoxy-pyrroloquinoline, was tested on a number of proteins. Only the expected product was obtained for the known quinoproteins, in a quantitative yield, as revealed by comparison with the values determined with the hydrazine method. Thus this independent method confirmed that bovine serum amine oxidase, porcine kidney diamine oxidase, dopamine /I-hydroxylase from bovine adrenal medulla, methylamine dehydrogenase from Thiobncillus versutus, glutamate decarboxylase from Escherichiu coli, and 3,4dihydroxyphenylalanine decarboxylase from pig kidney are really quinoproteins. Quantitative conversion was also achieved for condensation and addition products of PQQ (PQQ-acetone, PQQH,, PQQ-oxaxole, PQQ-dinitrophenylhydraxone, and PQQ-tryptophan). In view of this conversion and the fact that catalytic activity of PQQ is not required, the method seems suited to investigate the distribution of the cofactor in eukaryotes, especially in mammals where it is almost certain that PQQ occurs only in derivatized form. Finally, just like the hydrazine method, the hexanol extraction procedure seems unable to keep the structure of the cofactor as it exists in the active site, intact, as demonstrated for the pro-PQQ cofactor of methylamine dehydrogenase.
Extracellular laccase (benzenediol:oxygen oxidoreductase EC 1.10.3.2) from the lignin‐degrading fungus, Phlebia radiata, was shown to contain a novel combination of electron carriers as its prosthetic groups. In addition to two copper atoms per enzyme molecule, one molecule of PQQ was included as a cofactor. The EPR spectrum exhibits features of type 1 and type 2 copper atoms. In the enzymatic reaction 4 molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. During the reaction coniferyl alcohol is transformed to dilignols.
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(S)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 'H-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.
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