Ito N, Phillips S E, Stevens C, Ogel Z B, McPherson M J, Keen J N, Yadav K D, Knowles P F
Department of Biochemistry and Molecular Biology, University of Leeds, UK.
Nature. 1991 Mar 7;350(6313):87-90. doi: 10.1038/350087a0.
Galactose oxidase is an extracellular enzyme secreted by the fungus Dactylium dendroides. It is monomeric, with a relative molecular mass of 68,000, catalyses the stereospecific oxidation of a broad range of primary alcohol substrates and possesses a unique mononuclear copper site essential for catalysing a two-electron transfer reaction during the oxidation of primary alcohols to corresponding aldehydes. Recent evidence arguing against a Cu(III)-Cu(I) couple implies the existence of a second redox-active site proposed to involve pyrroloquinoline quinone or a tyrosine radical. We now report the crystal structure of galactose oxidase at 1.7 A resolution. This reveals a unique structural feature at the copper site with a novel thioether bond linking Cys 228 and Tyr 272 in a stacking interaction with Trp 290. We propose that these molecular components stabilize the protein free-radical species essential for catalysis and thus provide a 'built-in' secondary cofactor. This feature may represent a new mechanism for mediating electron transfer in metalloenzymes in the absence of exogenous cofactors.
半乳糖氧化酶是一种由树状指孢囊菌分泌的胞外酶。它是单体，相对分子质量为68,000，催化多种伯醇底物的立体特异性氧化，并且拥有一个独特的单核铜位点，该位点对于在伯醇氧化为相应醛的过程中催化双电子转移反应至关重要。最近有证据反对Cu(III)-Cu(I)偶联，这意味着存在第二个氧化还原活性位点，该位点被认为涉及吡咯喹啉醌或酪氨酸自由基。我们现在报道了分辨率为1.7埃的半乳糖氧化酶晶体结构。这揭示了铜位点处的一个独特结构特征，即通过一个新的硫醚键将半胱氨酸228和酪氨酸272连接起来，与色氨酸290形成堆积相互作用。我们提出这些分子组分稳定了催化所必需的蛋白质自由基物种，从而提供了一个“内置”的辅助因子。这一特征可能代表了在没有外源辅助因子的情况下金属酶中介导电子转移的一种新机制。
