Abstract
The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.
Highlights
The dependence of phytochelatin synthase (␥-glutamylcysteine dipeptidyltranspeptidase (PCS), EC 2.3.2.15) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme
AtPCS1 Is Constitutively Expressed in the Intact Plant—High stringency Northern analyses revealed a single 1.7-kb band of similar intensity after hybridization of random-primed 32P-labeled AtPCS1 cDNA with total RNA extracted from roots and shoots of 21-day-old Arabidopsis seedlings, regardless of whether the seedlings had been exposed to 25 or 100 M Cd2ϩ (CdSO4), the most potent activator of AtPCS1-catalyzed PC synthesis, Cu2ϩ (CuSO4), an activator of intermediate potency, or Zn2ϩ (ZnSO4), an activator of weak to moderate potency, for 6 h or for 24 h before RNA extraction (Fig. 1)
With the exception of Cu2ϩ, did pretreatment of the terminated reaction media with sodium borohydride before RP-HPLC markedly change the estimated reduced thiol contents of the PCs synthesized or the apparent rank order with which the metal cations or oxyanions promoted PC synthesis (Fig. 2), it was concluded that the effects of most of the metal cations and oxyanions examined were exerted at the enzyme level, not at the level of the oxidation state and amenability of the thiol peptide reaction products to detection with dithiobis(2-nitrobenzoic acid) (DTNB)
Summary
For PC synthase action, it has been proposed that the strongly conserved N-terminal half of the enzyme is responsible for catalysis and that activation arises from the binding of metal ions to residues, possibly cysteine residues, within this domain [4]. The presence of five conserved cysteine residues, two of which are vicinal, in the N-terminal halves of AtPCS1, SpPCS, and TaPCS1 is at least consistent with this notion, as is the observation that the three most extreme Arabidopsis cad alleles have amino acid substitutions in this region [12] An extension of this model, proposed to ascribe a role to the more sequence-divergent C-terminal half of the molecule and to account for the properties of the least extreme cad allele, cad, a nonsense mutation causing premature termination and deletion of the C-terminal segment, is the concept of a C-terminal “metal-sensing domain” whose multiple cysteine residues bind heavy metals and bring them into contact with the putative “activation” site within the N-terminal, catalytic half of the molecule. If heavy metals do directly bind AtPCS1 in vivo it is to a limited extent and associated with only minor augmentation of synthetic activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
