Oubrie A, Rozeboom H J, Kalk K H, Olsthoorn A J, Duine J A, Dijkstra B W
Laboratory of Biophysical Chemistry and BIOSON Research Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen.
EMBO J. 1999 Oct 1;18(19):5187-94. doi: 10.1093/emboj/18.19.5187.
Soluble glucose dehydrogenase (s-GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism of PQQ-dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X-ray structure of s-GDH with the cofactor at 2.2 A resolution, and of a complex with reduced PQQ and glucose at 1.9 A resolution. These structures reveal the active site of s-GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ-dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base-catalyzed hydride transfer, rather than the generally accepted covalent addition-elimination mechanism. Thus, PQQ-dependent enzymes use a mechanism similar to that of nicotinamide- and flavin-dependent oxidoreductases.
可溶性葡萄糖脱氢酶（s-GDH；EC 1.1.99.17）是一种典型的醌蛋白，它需要辅因子吡咯喹啉醌（PQQ）将葡萄糖氧化为葡萄糖酸内酯。由于缺乏全面的结构数据，PQQ依赖性酶的反应机制一直存在争议。我们已经确定了与辅因子结合的s-GDH的X射线结构，分辨率为2.2埃，以及与还原型PQQ和葡萄糖形成的复合物的结构，分辨率为1.9埃。这些结构揭示了s-GDH的活性位点，并首次展示了功能结合的底物如何在PQQ依赖性酶中与辅因子相互作用。在发现PQQ二十年后，我们的结果最终为一种包含一般碱催化氢化物转移的反应机制提供了确凿证据，而不是普遍接受的共价加成-消除机制。因此，PQQ依赖性酶使用的机制类似于烟酰胺和黄素依赖性氧化还原酶的机制。
