Poster: Heavy metals & phytoremediation

Abs # 54: Catalytic mechanism of phytochelatin synthase, a cysteine protease-like transpeptidase

Presenter:	Rea, Philip A., parea@sas.upenn.edu
Authors	Rea, Philip A. (A)   Vatamaniuk, Olena K. (A)   Lang, Albert  (A)   Mari, Stephane  (A)   Rigden, Daniel J. (B)
Affiliations:	(A): Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA (B): School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK

Phytochelatin (PC) synthases catalyze the synthesis of PCs, peptides competent in the high-affinity binding of heavy metals, from glutathione (GSH) and/or from previously synthesized PCs. Here we show by analyses of the stoichiometry of recombinant PC synthase-catalyzed PC synthesis, the kinetics and stoichiometry of acylation of the enzyme and the release of free glycine from γ-Glu-Cys donors, and the effects of the point substitution of conserved Cys and Ser residues that PC synthase is a dipeptidyl transferase that undergoes acylation at two sites, one of which likely corresponds to or is at least tightly coupled with C56. The identity of the second site of modification remains to be determined, but it is distinguishable from the first, which is amenable to acylation by free GSH, because its acylation not only depends on the provision of Cd or GSH with a blocked thiol group but is also necessary for net PC synthesis. It is concluded that the enzyme catalyzes a dipeptidyl transfer reaction in which some of the energy liberated upon cleavage of the Cys-Gly bonds of the γ-Glu-Cys donors in the first phase of the catalytic cycle is conserved through the formation of a two site substituted enzyme γ-Glu-Cys acyl intermediate whose hydrolysis in the second phase of the cycle provides the energy for formation of the new peptide bond required for PC chain extension. On the basis of these results and those from singly substituting the conserved D, H, T and Y residues in the catalytic half of the protein on activity, in combination with sequence comparison and validated three-dimensional model building, it is inferred that PC synthase is evolutionarily and mechanistically related to a specific clan of cysteine proteases to which papain also belongs.