Wachter R M, Branchaud B P
Department of Chemistry, University of Oregon, Eugene 97403, USA.
Biochemistry. 1996 Nov 12;35(45):14425-35. doi: 10.1021/bi961369x.
Galactose oxidase, a mononuclear copper enzyme, oxidizes primary alcohols to aldehydes using molecular oxygen. A unique type of cross-link between tyrosine 272, an active-site copper ligand, and cysteine 228 provides a modified tyrosine radical site believed to act as a one-electron redox center. Substrate analogs incorporating a primary thiol group in place of the primary alcohol group in normal substrates (RCH2OH) have been studied as active-site mechanistic probes. Thiol sulfur coordinates to the active-site copper, leading to enzyme inactivation in a time- and concentration-dependent manner. The mechanism of inactivation involves redox chemistry related to the active-site redox centers, though inactivation does not proceed through the rate-determining hydrogen atom abstraction step that occurs in alcohol oxidation. Thiols are therefore classified as active-site-directed redox inactivators. The thiol analog of galactose, 6-Thio-Me-Gal, is also turned over by the enzyme, albeit at a much reduced rate, indicating that the energetics of turnover is changed significantly. Thiols constitute a particularly good model of the ground state enzyme-substrate complex. The Michaelis complex for thiol substrate analogs is stabilized at least 200-fold compared to the analogous alcohol substrates, whereas the transition state of H atom abstraction is destabilized, presumably due to a slight increase in distances of reacting atoms and weakening of hydrogen-bonding interactions due to the larger atomic radius of sulfur compared to that of oxygen.
半乳糖氧化酶是一种单核铜酶，利用分子氧将伯醇氧化为醛。活性位点铜配体酪氨酸272与半胱氨酸228之间独特的交联方式形成了一个修饰的酪氨酸自由基位点，该位点被认为是一个单电子氧化还原中心。已将在正常底物（RCH2OH）中用伯硫醇基团取代伯醇基团的底物类似物作为活性位点机制探针进行了研究。硫醇硫与活性位点铜配位，导致酶以时间和浓度依赖性方式失活。失活机制涉及与活性位点氧化还原中心相关的氧化还原化学，尽管失活不是通过醇氧化中发生的速率决定步骤——氢原子提取进行的。因此，硫醇被归类为活性位点定向氧化还原失活剂。半乳糖的硫醇类似物6-硫代甲基半乳糖也能被该酶催化周转，尽管速率大大降低，这表明周转的能量学发生了显著变化。硫醇构成了基态酶-底物复合物的一个特别好的模型。与类似的醇底物相比，硫醇底物类似物的米氏复合物稳定至少200倍，而氢原子提取的过渡态则不稳定，这可能是由于反应原子间距离略有增加以及与氧相比硫的原子半径较大导致氢键相互作用减弱。
