Department of Chemistry, Stanford University, Stanford, California 94305, United States.
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 2, 166 10 Praha 6, Czech Republic.
J Am Chem Soc. 2023 Oct 25;145(42):22866-22870. doi: 10.1021/jacs.3c07450. Epub 2023 Oct 16.
Tyrosinase is a ubiquitous coupled binuclear copper enzyme that activates O toward the regioselective monooxygenation of monophenols to catechols via a mechanism that remains only partially defined. Here, we present new mechanistic insights into the initial steps of this monooxygenation reaction by employing a pre-steady-state, stopped-flow kinetics approach that allows for the direct measurement of the monooxygenation rates for a series of -substituted monophenols by oxy-tyrosinase. The obtained biphasic Hammett plot and the associated solvent kinetic isotope effect values provide direct evidence for an initial H-transfer from the protonated phenolic substrate to the CuO core of oxy-tyrosinase. The correlation of these experimental results to quantum mechanics/molecular mechanics calculations provides a detailed mechanistic description of this H-transfer step. These new mechanistic insights revise and expand our fundamental understanding of CuO active sites in biology.
酪氨酸酶是一种普遍存在的偶联双核铜酶，通过一种部分定义的机制，将 O 向单酚的区域选择性单加氧酶转化为儿茶酚。在这里，我们通过使用预稳态、停流动力学方法，对该单加氧反应的初始步骤提出了新的见解，该方法允许通过氧-酪氨酸酶直接测量一系列 -取代的单酚的单加氧酶速率。获得的双相哈梅特图和相关的溶剂动力学同位素效应值为从质子化的酚底物到氧-酪氨酸酶的 CuO 核心的初始 H 转移提供了直接证据。这些实验结果与量子力学/分子力学计算的相关性提供了对这个 H 转移步骤的详细的机制描述。这些新的见解修正并扩展了我们对生物中 CuO 活性中心的基本认识。
