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

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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.