Kay Christopher W M, Mennenga Bina, Görisch Helmut, Bittl Robert
Institut für Experimentalphysik, Fachbereich Physik, Freie Universität Berlin, 14195 Berlin, Germany.
J Am Chem Soc. 2005 Jun 8;127(22):7974-5. doi: 10.1021/ja050972c.
Pyrroloquinoline quinone (2,7,9-tricarboxypyrroloquinoline quinone, PQQ) is one of several quinone cofactors that is utilized in a class of dehydrogenases known as quinoproteins. In this contribution, we have used continuous-wave high-field/high-frequency electron paramagnetic resonance (EPR) at 94 GHz (W-band) to study substrate binding in ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa, taking advantage of the fact that the enzyme is isolated with a substantial proportion of the PQQ cofactor in the paramagnetic semiquinone form. In the substrate-free enzyme, the principal values of the g-tensor, obtained by spectral simulation are: gx = 2.00585(2), gy = 2.00518(2), and gz = 2.00212(2), giving giso = 2.00438(2). All three principal values of the g-tensor decrease when ethanol is bound to the protein: gx = 2.00574(2), gy = 2.00511(2), and gz = 2.00207(2), giving giso = 2.00431(2). The results represent the first direct evidence for the tight binding of an alcohol to a PQQ-dependent alcohol dehydrogenase and show that ethanol also binds to the enzyme even when the PQQ cofactor is in the semiquinone form. The decrease in g is consistent with an increase in polarity in the immediate vicinity of the PQQ cofactor and probably reflects a changed geometry of the PQQ-Ca2+ complex when ethanol binds.
吡咯喹啉醌（2,7,9 - 三羧基吡咯喹啉醌，PQQ）是一类被称为醌蛋白的脱氢酶中所使用的几种醌辅因子之一。在本论文中，我们利用94 GHz（W波段）的连续波高场/高频电子顺磁共振（EPR）来研究铜绿假单胞菌乙醇脱氢酶（QEDH）中的底物结合情况，这是因为该酶分离时含有相当比例的处于顺磁半醌形式的PQQ辅因子。在无底物的酶中，通过光谱模拟得到的g张量主值为：gx = 2.00585(2)，gy = 2.00518(2)，gz = 2.00212(2)，giso = 2.00438(2)。当乙醇与蛋白质结合时，g张量的所有三个主值均减小：gx = 2.00574(2)，gy = 2.00511(2)，gz = 2.00207(2)，giso = 2.00431(2)。这些结果代表了醇类与PQQ依赖的醇脱氢酶紧密结合的首个直接证据，并表明即使PQQ辅因子处于半醌形式，乙醇也能与该酶结合。g值的降低与PQQ辅因子紧邻区域极性的增加相一致，可能反映了乙醇结合时PQQ - Ca2+复合物几何结构的变化。
