Leufken Christine M, Moerschbacher Bruno M, Dirks-Hofmeister Mareike E
Institute of Plant Biology and Biotechnology, Westphalian Wilhelm's-University of Münster, Schlossplatz 8, 48143 Münster, Germany.
Biochim Biophys Acta. 2015 Feb;1854(2):178-86. doi: 10.1016/j.bbapap.2014.11.007. Epub 2014 Dec 5.
Plant polyphenol oxidases (PPOs) have a conserved three-domain structure: (i) the N-terminal domain (containing the active site) is connected via (ii) a linker to (iii) the C-terminal domain. The latter covers the active site, thereby maintaining the enzyme in a latent state. Activation can be achieved with SDS but little is known about the mechanism. We prepared domain-swap variants of dandelion PPO-1 and PPO-2 to test the specific functions of individual domains and their impact on enzyme characteristics. Our experiments revealed that the C-terminal domain modulates the pH optimum curve and has a strong influence on the optimal pH value. The linker determines the SDS concentration required for full activation. It also influences the SDS concentration required for half maximal activation (kSDS) and the stability of the enzyme during prolonged incubation in buffers containing SDS, but the N-terminal domain has the strongest effect on these parameters. The N-terminal domain also determines the IC50 of SDS and the stability in buffers containing or lacking SDS. We propose that the linker and C-terminal domain fine-tune the activation of plant PPOs. The C-terminal domain adjusts the pH optimum and the linker probably contains an SDS-binding/interaction site that influences inactivation and determines the SDS concentration required for activation. For the first time, we have determined the influence of the three PPO domains on enzyme activation and stability providing insight into the regulation and activation mechanisms of type-3 copper proteins in general.
植物多酚氧化酶（PPOs）具有保守的三结构域结构：（i）N端结构域（包含活性位点）通过（ii）一个连接子与（iii）C端结构域相连。后者覆盖活性位点，从而使酶处于潜伏状态。用SDS可实现激活，但对其机制了解甚少。我们制备了蒲公英PPO - 1和PPO - 2的结构域交换变体，以测试各个结构域的特定功能及其对酶特性的影响。我们的实验表明，C端结构域调节最适pH曲线，并对最适pH值有很大影响。连接子决定了完全激活所需的SDS浓度。它还影响半最大激活所需的SDS浓度（kSDS）以及在含有SDS的缓冲液中长时间孵育期间酶的稳定性，但N端结构域对这些参数的影响最强。N端结构域还决定了SDS的IC50以及在含有或缺乏SDS的缓冲液中的稳定性。我们提出，连接子和C端结构域对植物PPOs的激活进行微调。C端结构域调节最适pH，连接子可能含有一个SDS结合/相互作用位点，该位点影响失活并决定激活所需的SDS浓度。我们首次确定了PPO的三个结构域对酶激活和稳定性的影响，为一般3型铜蛋白的调节和激活机制提供了深入了解。
