**2. The biochemical mechanism of phytochelatin synthase**

#### **2.1 The domain organization of phytochelatin synthase**

The eukaryotic PCS has two domains with distinct functions: a conserved N-terminal domain that shows γ-glutamylcysteine dipeptidyl transpeptidase activity and a variable C-terminal domain involved in metal sensing [52–54]. Using AtPCS1 as a model, the molecular functions of the N- and C-domains as well as the catalytic mechanism of eukaryotic PCS were revealed [6, 53–55]. The N-terminal half AtPCS1 is sufficient for deglycination of GSH and elongating PC molecules, indicating that the N-terminal domain carries out the core catalysis [53, 56]. However, the truncated AtPCS1 without the C-terminal domain is less thermostable and has lower PC synthetic activity than the full-length enzyme [53, 55, 56]. Notably, the deletion of the C-terminal domain completely impairs the PC synthesis activity of the enzyme in the presence of Zn2+ and partially inactivates PC synthesis in the Cd- or Hg-containing reactions [53, 55]. These findings suggest that the C-terminal domain is essential for stabilizing the protein and functions as a metal sensor [53, 55]. More evidence has shown that the C-terminal end of AtPCS1 is required for the augmentation of PC synthetic activity. One example is that the residues from Asp373 to the C-terminal end of AtPCS1 contain multiple regions involved in Zn-dependent and As-dependent activation of PC synthesis [45, 57, 58]. (Also see Section 2.4: The activation of phytochelatin synthase through the chelation of heavy metal ions).

PCS-like sequences also exist in prokaryotes with moderate sequence homology to the N-domain of eukaryotic PCS [39–41]. However, prokaryotic PCS likely has unique functions apart from PC synthesis. For example, the PCS homolog found in cyanobacterium *Nostoc* sp. PCC 7120 (NsPCS) shows distinct characters from its eukaryotic counterparts that efficiently catalyze PC synthesis. NsPCS is a "half-PCS molecule" that does not have a C-terminal domain [39, 40, 52] and catalyzes the hydrolysis of GSH at a high rate and the synthesis of PCs at a relatively low rate [39, 40, 42]. Besides, the enzyme activity of NsPCS seems insensitive to the absence or presence of Cd2+, which suggest that the prokaryotic PCS is involved in GSH metabolism in the cells rather than the responses to heavy metal stress [39, 42].

#### **2.2 The core catalytic mechanism**

Vatamaniuk et al. [6] first confirmed that the synthesis of PCs occurs through a ping-pong mechanism and involves two substrates: one GSH as the low-affinity substrate for the first step of PC synthesis and one metal-GSH conjugate as the highaffinity substrate for the second step [6]. In the standard PC synthesis reactions *in vitro*, which resemble the concentrations of the GSH and metal ions in the cytosol, GSH exists at a considerably higher level (millimolar) than heavy metal ions (micromolar) [6, 7, 59]. Presumably, more than 98% of total metal ions in this condition are associated with GSH as bis(glutathionato)metal ions (metal∙GS2), and the free Cd concentration can be as low as 10−6 μM [6]. Under these circumstances, GSH and Cd∙GS2 are two separate compounds for PC synthesis.

Right after GSH enters the catalytic site of PCS, a Gly residue is removed to form the γGlu-Cys acyl-enzyme intermediate, and then a metal-GS2 accepts the γGlu-Cys unit to generate a PC2 ((γGlu-Cys)2-Gly) [6, 21, 54, 55]. Following the synthesis of PC2, the elongation of PCs occurs using previously synthesized PCs as acceptors to receive γGlu-Cys [6, 21, 54, 55, 60]. The whole process can be described as two equations:

$$\text{PCS} + \gamma \text{Glu\text{\textquotedblleft}C} \text{ys\textquotedblright} \text{-} \text{Gly} \rightarrow \text{PCS} \text{ -} \gamma \text{Glu\textquotedblright} \text{-} \text{Cys} + \text{Gly} \tag{1}$$

$$\begin{array}{l}\text{PCS - }\gamma\text{Glu - Cys + metal} \cdot \left(\gamma\text{Glu - Cys -Gly}\right)\_2 \rightarrow \text{PCS} \\ + \text{metal} \cdot \left(\gamma\text{Glu - Cys}\right)\_2 \cdot \text{Gly} + \gamma\text{Glu - Cys -Gly} \end{array} \tag{2}$$

Overall, PCS catalyzes the peptide chain elongation from C-to-N terminus [4, 6, 21, 54]:

$$\begin{array}{l} \left(\gamma \text{Glu\text{ -}Cys\text{)} - Gly + \left(\gamma \text{Glu\text{ -}Cys\text{)}}\_{u} \text{ -} Gly\right) \\ \rightarrow \left(\gamma \text{Glu\text{ -}Cys\text{)} \cdot \left(\gamma \text{Glu\text{ -}Cys\text{)}}\_{u} \text{ -} Gly + Gly\right) \end{array} \tag{3}$$

PCS and Cys proteases share similar core catalytic mechanisms to hydrolyze a GSH molecule and form a γ-Glu-Cys-acyl–enzyme intermediate [41, 52]. The Cys protease-like catalytic triad of PCS was confirmed based on the mutagenic studies of AtPCS1 and the crystal structures of NsPCS [41, 54, 55]. Vatamaniuk et al. and Romanyuk et al. reported that Cys56, His162, and Asp180 of AtPCS1 are the three residues of the catalytic triad among divergent PCS sequences [54, 55]. The molecular interaction between these residues and GSH was further revealed by Vivares et al. with the crystal structures of native NsPCS and the γ-Glu-Cys-acyl–enzyme intermediate at a 2.0-Å resolution [41]. Although NsPCS only shares 36% identity at the amino acid level with AtPCS1, it contains the conserved catalytic triad and can catalyze the deglycination of GSH [39–42]. These crystal structures provide details about the hydrolysis of the peptide bond that involves Cys56 and the 3D structure of the Cys-His-Asp catalytic triad [41]. It is worth mentioning that NsPCS formed homodimers in the crystallization experiments [26]. This is in agreement with the dimerization of the partially purified PCS from *Silene cucubalus*, which was confirmed by determining the native molecular weight of the protein [4].

#### **2.3 Critical amino acids contributing to the enzyme activity**

Based on the high-resolution crystal structure of NsPCS, multiple research groups have simulated putative 3D structures of eukaryotic PCS using various programs, and these structure models provide valuable information that uncovers

#### *Phytochelatin Synthase in Heavy Metal Detoxification and Xenobiotic Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99077*

the conserved molecular mechanism of PCS [56, 61–65]. For example, the molecular models of AtPCS1 reveal the key amino acids that contribute to the mechanism of the second substrate recognition and the enzyme activation through Thr phosphorylation [56, 61]. Chia et al. first reported how AtPCS1 might attract and stabilize the second substrate, metal-GS2, after the γ-Glu-Cys-acyl–enzyme intermediate is formed [61]. In this study, the modeled AtPCS1 structure revealed a pocket in proximity to the first substrate-binding site, consisting of three loops containing several conserved amino acids, including Arg152, Lys185, and Tyr55. Mutations on Arg152 or Lys185 (Arg-to-Lys or Lys-to-Arg substitutions) resulted in the complete abrogation of enzyme activity, indicating that the arrangement of these positive charges is crucial for the binding of the second substrate. Mutations at Tyr55 did not completely impair the enzyme activity, but the Tyr55 mutant AtPCS1 showed lower catalytic activities than the wild-type enzyme due to a reduced affinity to metal-GS2. In addition, the mutation at Tyr55 reduced Cd2+ binding ability of the AtPCS1 protein. It was therefore suggested that Tyr55 binds to the Cd ion of metal-GS2 through cation-π interaction and thus contributes to the recognition of the second substrate. Besides these three amino acids, other conserved residues on the loops constituting the second substrate-binding pocket, including Gln50, Glu52, Glen157, Phe184, and Tyr186, are also important for the PC synthesis activity [61, 62].

Wang et al. identified that Thr49 is the phosphorylation site related to the activation of AtPCS1 [56]. The mutant AtPCS1 with Thr49-to-Ala49 substitution could not be phosphorylated, and its PC synthesis activity was significantly lower than that of the wild-type enzyme. According to the proposed 3D model of AtPCS1, Thr49 is within proximity to Arg183, which is also crucial for the catalytic activity of AtPCS1, and both residues are next to the catalytic site and substrate binding pockets. It was proposed that the phosphorylated Thr49 interacts with Arg183, and that this interaction serves as a "molecular clip" to give the active site a conformation appropriate for catalysis. Because Thr49 and Arg183 are both highly conserved among PCS sequences, the activity of eukaryotic PCS may as well be regulated by similar phosphorylation modifications [56, 66].

### **2.4 The activation of phytochelatin synthase through the chelation of heavy metal ions**

As a key component of early response to heavy metal stress, PCS protein is constitutively expressed in the cytosol for rapid activation stimulated by heavy metals [2, 3, 6]. The heavy metal ions entering cytosol are essential for forming the second substrate [6, 60]. They can also bind to PCS, resulting in augmentative activation [6, 55, 67, 68]. Moreover, heavy metals could be a critical factor that triggers PCS phosphorylation [56, 69]. For example, AtPCS1 phosphorylation only occurred in the presence of Cd2+ in the *in vitro* experiments [56].

PCS, confirmed to be a metalloenzyme *in vitro*, is also likely to be one *in vivo* [6, 17]. Equilibrium analyses show that one AtPCS1 molecule binds seven Cd2+ in solutions containing 10 μM CdCl2 [6, 61]. Apart from Tyr55, which is proposed to bind the Cd2+ on the metal-GS2, the Cd binding capability of PCS presumably comes from conserved Cys pairs and CysXXCys motifs also found in metallothionein [30, 61]. Peptide screening of SpPCS and TaPCS showed that the core sequences containing consensus Cys-rich motifs could bind Cd2+ *in vitro* [67]. The subsequent site-direct mutagenesis analysis indicated that conserved Cys pairs at the N-terminal domain were critical for PCS activity, while the Cys-rich motifs at the C-terminal domain only slightly affected the PC synthesis rate [67]. It is not yet clear how these

Cys-rich motifs enhance the PC synthesis rate. It is possible that they bind metal-GS2 complexes or free metal ions to stabilize the protein structure [6, 30, 55, 67]. More investigations are still needed to explain the molecular functions of these Cys-rich motifs and how they participate in the metal activation of PCS.
