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.