Fluorescence Spectroscopy Investigation of the Enzymatic Mechanism of Phytochelatin Synthase, an Enzyme Able to Cleave and Form Peptide Bond

植物螯合素合酶（PCS）由谷胱甘肽（GSH）形成植物螯合素（PC）。PC是植物重金属解毒所必需的。然而，在许多细菌和一些动物物种中也发现了PCS基因。PCS的催化机制可以分为两部分：（i）肽酶活性：从GSH裂解甘氨酸，这导致形成共价结合的γ-谷氨酰-半胱氨酸二肽;（ii）转肽酶活性：在所得γ-谷氨酰-半胱氨酸二肽和另一个GSH分子之间形成肽键。虽然一些生理学研究调查PC合成的功能，没有详细的生物物理研究的机制，PCS已被执行。该项目旨在通过表征催化循环的能量学和动力学来阐明PCS的分子机理。为此，我们希望采用荧光标记和化学修饰的GSH类似物。缺乏转肽酶活性的细菌PCS样酶和植物PCS显示这两种活动之间的比较将提供这些活动的决定因素的信息。通过实验（荧光，紫外-可见和核磁共振光谱）与理论（分子动力学，静电学，量子化学）技术相结合，我们希望研究PCS的功能，并将结构信息与生理数据联系起来。

Phytochelatin synthase (PCS) forms phytochelatin (PC) from glutathione (GSH). PCs are known to be essential for the heavy metal detoxification in plants. However, the gene of PCS has been also found in many bacteria and several animal species. The catalytic mechanism of PCS can be divided in two parts: (i) peptidase activity: the cleavage of a glycine from GSH, which leads to the formation of a covalently-bound γ-glutamyl-cysteine dipeptide (ii) transpeptidase activity: the formation of a peptide bond between the resulting γ-glutamyl-cysteine dipeptide and another GSH molecule. Although several physiological studies investigate the function of PC synthesis, no detailed biophysical investigation of the mechanism of PCS has been performed yet. The proposed project aims to elucidate the molecular mechanism of PCS by characterizing the energetics and the kinetics of the catalytic cycle. For this purpose, we want to employ fluorescently-labeled and chemically-modified GSH analogues. The comparison between bacterial PCS-like enzymes lacking the transpeptidase activity and plant PCS displaying both activities will provide information on the determinants of these activities. By combining experimental (Fluorescence, UV-Vis and NMR spectroscopy) with theoretical (molecular dynamics, electrostatics, quantum chemistry) techniques, we want to investigate the function of PCS and to connect the structural information with physiological data.