Phytochelatin (PC) synthases are -glutamylcysteine dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (-Glu-Cys)_nGly polymers, from glutathione (GSH) and/or shorter-chain PCs. Here it is shown through investigations of the enzyme from 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 Cd²⁺. Purified N-terminally hexahistidinyl-tagged 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 Cd²⁺ 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 -Glu-Cys acylation at two sites during the Cd²⁺-dependent synthesis of PCs from GSH and is stimulated by free Cd²⁺ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes -Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated but instead inhibited by free Cd²⁺ 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 (Rea, P.A., Vatamaniuk, O.K., and Rigden, D.J. (2004) Plant Physiol., 136: 2463-2474), it has been determined that the N-terminal half of AtPCS1, and its equivalents from other sources, has the hallmarks of a papain-like, Clan CA, cysteine protease. While 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 (Vivares, D., Arnoux, P., and Pignol, D. (2005) Proc. Natl. Acad. Sci. USA, 102: 18848-18853, Rea, P.A. (2006) Proc. Natl. Acad. Sci. USA, 103: 507-508), is capable of explaining the strict requirement for a conserved Cys residue, Cys⁵⁶ in the case of AtPCS1, for formation of the biosynthetically competent -Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining if the other two residues, His¹⁶² and Asp¹⁸⁰, 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 which demonstrate that not only Cys⁵⁶ but also His¹⁶² and Asp¹⁸⁰ are indeed required for net PC synthesis. It is therefore established experimentally for the first time that AtPCS1, and by implication other eukaryotic PC synthases, are papain cysteine protease superfamily members but ones, unlike their prokaryotic counterparts, which in addition to having a papain-like N-terminal catalytic domain that undergoes primary -Glu-Cys acylation contain an auxiliary metal-sensing C-terminal domain that undergoes secondary -Glu-Cys acylation.