Shank M, Barynin V, Dismukes G C
Department of Chemistry, Hoyt Laboratory, Princeton University, New Jersey 08544-1009.
Biochemistry. 1994 Dec 27;33(51):15433-6. doi: 10.1021/bi00255a025.
Catalysis of hydrogen peroxide dismutation by the dimanganese catalase from Thermus thermophilus has been measured and found to obey Michaelis-Menton kinetics with no evidence for substrate inhibition at concentrations up to 0.45 M H2O2. Comparison among three dimanganese catalases (Thermus thermophilus, Thermoleophilium album, and Lactobacillus plantarum) reveals that their apparent second-order rate constants, Kcat/Km, differ by at most a factor of 5, even though the individual kinetic constants differ by as much as a factor of 20. This similarity suggests that all three enzymes may have the same rate-determining step. For T. thermophilus catalase we find that kcat/Km approximately kbi, the bimolecular rate constant at limiting substrate concentrations. Thus, the rate of the rate-determining step is unaltered over the entire range of substrate concentrations, unlike T. album and L. plantarum catalases where substrate inhibition has been reported. Comparison to structurally characterized dimanganese complexes and dimetalloproteins (arginase, hemerythrin), which are functional, albeit kinetically slow, catalase mimics, reveals that high catalase activity correlates with a greater number of stronger sigma-ligand donors like anionic carboxylatos vs neutral histidines that stabilize the oxidized Mn2(III,III) state over reduced Mn2(II,II). A critical feature for enzymatic functionality in vivo is suppression of one-electron chemistry leading to formation of the mixed-valence forms, Mn2(III,IV) and Mn2(II,III), which are kinetically inactive or precursors to inactive species, respectively. Evidence is presented from model compounds suggesting that the mu-carboxylato bridge between Mn ions in catalase may play the key role in suppressing formation of these detrimental oxidation states through destabilization of these one-electron redox processes.
嗜热栖热菌双锰过氧化氢酶催化过氧化氢歧化反应的活性已被测定，结果发现其遵循米氏动力学，在过氧化氢浓度高达0.45 M时未发现底物抑制现象。对三种双锰过氧化氢酶（嗜热栖热菌、嗜热栖热嗜热栖热菌和植物乳杆菌）的比较表明，尽管它们各自的动力学常数相差高达20倍，但其表观二级速率常数Kcat/Km最多相差5倍。这种相似性表明，这三种酶可能具有相同的速率决定步骤。对于嗜热栖热菌过氧化氢酶，我们发现kcat/Km约等于kbi，即极限底物浓度下的双分子速率常数。因此，与报道存在底物抑制现象的嗜热栖热嗜热栖热菌和植物乳杆菌过氧化氢酶不同，速率决定步骤的速率在整个底物浓度范围内保持不变。与结构已明确的双锰配合物和双金属蛋白（精氨酸酶、蚯蚓血红蛋白）进行比较，这些物质虽然在动力学上较慢，但具有过氧化氢酶模拟功能，结果表明，高过氧化氢酶活性与更多更强的σ-配体供体相关，如阴离子羧酸盐与中性组氨酸，它们能使氧化态的Mn2(III,III)比还原态的Mn2(II,II)更稳定。体内酶功能的一个关键特征是抑制单电子化学反应，从而避免形成混合价态形式Mn2(III,IV)和Mn2(II,III)，它们分别在动力学上无活性或为无活性物种的前体。模型化合物提供的证据表明，过氧化氢酶中锰离子之间的μ-羧酸盐桥可能通过破坏这些单电子氧化还原过程的稳定性，在抑制这些有害氧化态的形成中起关键作用。
