Phytochelatins are small enzyme-synthesized peptides that mediate tolerance to several heavy metals. To gain insight into the unusual metal catalytic site of the Euglena gracilis phytochelatin synthase (EgPCS), site-directed mutagenesis was performed on the C-terminal Cys/His (C/H) rich region 421-C-C-X-H-X-H-X-H-H-H-430. EgPCS mutants were analyzed at the cellular, kinetic and structural levels. Cd²⁺-resistance was conferred to Cd²⁺-sensitive yeast cells by EgPCS_wt (476aa) and truncated EgPCS_435 (435aa), but not by EgPCS_415 (415aa), suggesting that the resistance phenotype was linked to the enzyme C/H rich region. Moreover, full-length mutants (in which C and H residues in the C/H rich region were replaced by Ala) EgPCS_C2 (C421A/C422A) and EgPCS_C2H5 (C421A/C422A/H424A/H426A/H428A/H429A/H430A) did not provide Cd²⁺-resistance; in contrast, EgPCS_H2 (H424A/H426A), EgPCS_H3 (H428A/H429A/H430A), and EgPCS_H5 (H424A/H426A/H428A/H429A/H430A) mutants conferred similar Cd²⁺-tolerance like EgPCS_wt. Kinetic analysis showed that maximal rate (V_max) for PC₂ synthesis, affinity constants (K_{m Zn-GS2} or K_{m Cd-GS2}) and catalytic efficiencies (V_max/K_m) were differentially impaired in the mutants, as compared to EgPCS_wt, with EgPCS_C2 being the most perturbed enzyme; however, the K_0.5 values for GSH were not affected. All EgPCS mutants were predominantly monomeric. Far UV circular dichroism spectra and differential scanning calorimetry endotherms, indicated that alterations of the catalytic properties of EgPCS_C2 were not due to partial unfolding or destabilization of the native state of this mutant. The results indicated that C421/C422 and H424A/H426A/H428/H429/H430 are essential components of EgPCS for catalysis and activation by metal-substrate complexes.

植物螯合素是小酶合成的肽，介导对几种重金属的耐受性。为了深入了解细粒裸藻植物螯合酶合酶（Eg PCS）的异常金属催化位点，对富含C端Cys / His（C / H）的区域421-CCXHXHXHHH-430进行了定点诱变。例如，在细胞，动力学和结构水平上分析了PCS突变体。镉²⁺ -resistance被授予对Cd ²⁺通过敏感的酵母细胞例如PCS_wt（476aa）和截短的例如由PCS_435（435aa），但不例如PCS_415（415aa），表明抗性表型与酶C / H富集区相关。此外，全长突变体（其中富含C / H的区域中的C和H残基被Ala取代）例如PCS_C2（C421A / C422A）和例如PCS_C2H5（C421A / C422A / H424A / H426A / H428A / H429A / H430A）没有提供Cd ²⁺抗性；与此相反，例如PCS_H2（H424A / H426A），EG PCS_H3（H428A / H429A / H430A），和例如PCS_H5（H424A / H426A / H428A / H429A / H430A）突变体赋予类似镉²⁺公差的像例如PCS_wt。动力学分析表明，PC ₂合成的最大速率（V _max）为亲和常数（ķ_米的Zn-GS2或ķ_米镉GS2）和催化效率（V_毫安X / ķ_米）的差异在受损的突变体，相比于例如PCS_wt，与例如PCS_C2是最扰动酶; 但是，GSH的K _0.5值不受影响。所有的Eg PCS突变体主要是单体。远紫外圆二色性光谱和差示扫描量热法吸热表明，Eg的催化性能发生了变化PCS_C2不是由于该突变体的天然状态的部分展开或不稳定引起的。结果表明，C421 / C422和H424A / H426A / H428 / H429 / H430是Eg PCS用于金属-底物配合物催化和活化的必要成分。