Zhu Hui, Sommerhalter Monika, Nguy Andy K L, Klinman Judith P
#Department of Chemistry and Biochemistry, California State University, East Bay, 25800 Carlos Bee Boulevard, Hayward, California 94542, United States.
J Am Chem Soc. 2015 May 6;137(17):5720-9. doi: 10.1021/ja512388n. Epub 2015 Apr 28.
Tyramine β-monooxygenase (TβM) belongs to a family of physiologically important dinuclear copper monooxygenases that function with a solvent-exposed active site. To accomplish each enzymatic turnover, an electron transfer (ET) must occur between two solvent-separated copper centers. In wild-type TβM, this event is too fast to be rate limiting. However, we have recently shown [Osborne, R. L.; et al. Biochemistry 2013, 52, 1179] that the Tyr216Ala variant of TβM leads to rate-limiting ET. In this study, we present a pH-rate profile study of Tyr216Ala, together with deuterium oxide solvent kinetic isotope effects (KIEs). A solvent KIE of 2 on kcat is found in a region where kcat is pH/pD independent. As a control, the variant Tyr216Trp, for which ET is not rate determining, displays a solvent KIE of unity. We conclude, therefore, that the observed solvent KIE arises from the rate-limiting ET step in the Tyr216Ala variant, and show how small solvent KIEs (ca. 2) can be fully accommodated from equilibrium effects within the Marcus equation. To gain insight into the role of the enzyme in the long-range ET step, a temperature dependence study was also pursued. The small enthalpic barrier of ET (Ea = 3.6 kcal/mol) implicates a significant entropic barrier, which is attributed to the requirement for extensive rearrangement of the inter-copper environment during PCET catalyzed by the Tyr216Ala variant. The data lead to the proposal of a distinct inter-domain pathway for PCET in the dinuclear copper monooxygenases.
酪胺β-单加氧酶（TβM）属于一类具有重要生理功能的双核铜单加氧酶家族，其活性位点暴露于溶剂中。为了完成每一次酶促周转，必须在两个被溶剂隔开的铜中心之间发生电子转移（ET）。在野生型TβM中，这一过程太快，不是限速步骤。然而，我们最近发现[奥斯本，R.L.等人，《生物化学》，2013年，第52卷，第1179页]，TβM的Tyr216Ala变体导致ET成为限速步骤。在本研究中，我们展示了Tyr216Ala的pH-速率曲线研究以及重水溶剂动力学同位素效应（KIEs）。在kcat与pH/pD无关的区域发现kcat的溶剂KIE为2。作为对照，ET不是速率决定因素的Tyr216Trp变体显示溶剂KIE为1。因此，我们得出结论，观察到的溶剂KIE源于Tyr216Ala变体中的限速ET步骤，并展示了Marcus方程中的平衡效应如何完全解释小的溶剂KIE（约为2）。为了深入了解酶在远程ET步骤中的作用，我们还进行了温度依赖性研究。ET的小焓垒（Ea = 3.6千卡/摩尔）表明存在显著的熵垒，这归因于Tyr216Ala变体催化的质子耦合电子转移（PCET）过程中铜间环境需要广泛重排。这些数据促使我们提出了双核铜单加氧酶中PCET的独特域间途径。
