Determination of PQQ-adduct with PQQ-liberating enzyme

将PQQ与丝氨酸孵育形成两种PQQ-丝氨酸。这些PQQ-丝氨酸的比例取决于反应混合物的pH值。当在pH 8.0下与丝氨酸形成PQQ-加合物时，最简单的PQQ-恶唑-PQQ-甘氨酸是唯一的PQQ-加合物。另一方面，PQQ-加合物在pH为3.5的条件下形成另一种加合物。这两个PQQ加合物本身是稳定的，不能相互转化。结构分析表明，在pH值为3.5的条件下制备的PQQ-丝氨酸中含有丝氨酸来源的-CH2OH基团。当PQQ与各种氨基酸在pH 3.5下反应时，只生成一种与PQQ-甘氨酸化学结构不同的PQQ加合物。PQQ-丝氨酸和PQQ-丝氨酸对微生物都表现出明显的促生长活性，在特定的条件下，PQQ对大肠杆菌有明显的促生长作用。这种PQQ效应被归类为II型效应。当大肠杆菌在含有PQQ的培养基上生长时，大约50%的GDH在培养过程中被转化为全酶。在这一时刻，大肠杆菌消耗了大量的葡萄糖，而且培养基比不含PQQ的培养基酸化程度更高。根据这些结果，GDH将葡萄糖氧化成葡萄糖酸，但葡萄糖酸几乎没有被细胞吸收。GDH的功能是产生质子梯度，然后是ATP合成。三磷酸腺苷通过PTS支持葡萄糖的摄取，从而刺激大肠杆菌的生长。Apo-GDH在微生物中广泛分布，因此，这种PQQ效应被认为是一种普遍现象。讨论了PQQ和PQQ-加合物对微生物的促生长作用机理。

Two species of PQQ-serine were formed by incubating PQQ with serine. The ratio of these PQQ-serine was depend on pH of the reaction mixture. When PQQ-adducts formation with serine was conducted at pH 8.0, the simplest PQQ-oxazole, PQQ-glycine was given as sole PQQ-adduct. On the other hand, another species of PQQ-adduct was formed when incubated at pH 3.5. These two PQQ-adducts were stable themselves and were not interconvertible each other. As a result of structural analysis, the PQQ-serine prepared at pH 3.5 has a -CH_2OH group originated from serine. When the reaction of PQQ with various amino acids was carried out at pH 3.5, only one species of PQQ-adduct was formed, which are different in chemical structure from PQQ-glycine. Both species of PQQ-serine exhibited a marked growth stimulating activity for microorganisms.Growth stimulating effect of PQQ for E. coli was clearly observed under well defined conditions. This PQQ effect was classfied to the type II effect. When E. coli grew on a medium containing PQQ, about 50% of whole GDH was converted to holoenzyme during cultivation. At this moment, E. coli consumed a large amount of glucose and the medium was highly acidified more than in a medium excluding PQQ. From these results, GDH oxidizes glucose to gluconate but gluconate was scarecely taken up into the cell. GDH functions to generate proton gradient, followed by ATP synthesis. The ATP supports glucose uptake via PTS, subsequently the growth of E. coli is stimulated. Apo-GDH is widely distributed in microorganisms, thus, this PQQ effect is estimated to be an universal phenomenon. A proposed mechanism of growth stimulating effect of PQQ and PQQ-adducts for microorganism is discussed.