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pq -丝氨酸的比例取决于反应混合物的pH。在pH 8.0下与丝氨酸生成pqq -加合物时，给出了最简单的pqq -恶唑、pqq -甘氨酸作为唯一的pqq -加合物。另一方面，当pH为3.5时，形成了另一种pq -加合物。这两种pqq -加合物本身是稳定的，彼此之间不能相互转化。结构分析表明，在pH 3.5条件下制备的pq -丝氨酸含有一个源自丝氨酸的-CH_2OH基团。当PQQ与多种氨基酸在pH 3.5下反应时，只生成一种PQQ-加合物，其化学结构与PQQ-甘氨酸不同。两种pq -丝氨酸对微生物均表现出明显的促生长活性。在确定的条件下，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，整个GDH在培养过程中约有50%转化为全酶。此时，大肠杆菌消耗了大量葡萄糖，培养基的酸化程度高于不含PQQ的培养基。从这些结果来看，GDH将葡萄糖氧化为葡萄糖酸盐，但葡萄糖酸盐很少被吸收到细胞中。GDH的作用是产生质子梯度，然后合成ATP。ATP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效应估计是一种普遍现象。讨论了PQQ及其加合物对微生物的促生长作用机制。

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.