Phytochelatin (PC) synthases are g-glutamylcysteine (g-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (g-Glu-Cys) n Gly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd 21 . A purified N-terminally hexahistidinyltagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd 21 or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes g-Glu-Cys acylation at two sites during the Cd 21 -dependent synthesis of PCs from GSH and is stimulated by free Cd 21 when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes g-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd 21 when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent g-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary g-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary g-Glu-Cys acylation.

Phytochelatin (PC) synthase has been assumed to be a ␥-glutamylcysteine dipeptidyl transpeptidase (EC 2.3.2.15) and, more recently, as exemplified by analyses of the immunopurified recombinant enzyme from Arabidopsis thaliana (AtPCS1-FLAG), has been shown to catalyze a PC synthetic reaction with kinetics that approximates a bisubstrate-substituted enzyme mechanism in which millimolar concentrations of free GSH and micromolar concentrations of heavy metal⅐GSH thiolates (e.g. cadmium⅐GS 2 ) or millimolar concentrations of S-alkylglutathiones serve as cosubstrates. Here, we show, by direct analyses of the stoichiometry of AtPCS1-FLAG-catalyzed PC synthesis, the kinetics and stoichiometry of acylation of the enzyme and release of free glycine from ␥-Glu-Cys donors, and the effects of the Cys-to-Ser or -Ala and Serto-Ala substitution of conserved residues in the catalytic N-terminal half of the enzyme, that PC synthase is indeed a dipeptidyltransferase that undergoes ␥-Glu-Cys acylation at two sites during catalysis, one of which, in accord with a cysteine protease model, likely corresponds to or is at least tightly coupled with Cys 56. The identity of the second site of enzyme modification remains to be determined, but it is distinguishable from the first Cys 56 -dependent site, which is amenable to ␥-Glu-Cys acylation by free GSH, because its acylation not only depends on the provision of Cd 2؉ or GSH with a blocked, S-alkylated thiol group, but is also necessary for net PC synthesis. We conclude that des-Gly-PCs are not generated as an immediate by-product, but rather that the enzyme catalyzes a dipeptidyl transfer reaction in which some of the energy liberated upon cleavage of the Cys-Gly bonds of the ␥-Glu-Cys donors in the first phase of the catalytic cycle is conserved through the formation of a two site-substituted ␥-Glu-Cys acyl-enzyme intermediate whose hydrolysis provides the energy required for the formation of the new peptide bond required for the extension of PC chain length by one ␥-Glu-Cys repeat per catalytic cycle.

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.