Characterization of the acid/base and redox chemistry of phytochelatin analogue peptides.

The phytochelatins are a family of polydisperse, thiol-rich peptides that are synthesized by plants in response to exposure to heavy metals. The amino acid sequence of the phytochelatin peptides is (gamma-glutamyl-cysteinyl)n-glycine, where n typically ranges from 2 to 5. In the first phase of a program to characterize the coordination chemistry of the phytochelatins with heavy metals, the phytochelatin analogue peptides acetyl(gamma-glutamyl-cysteinyl)n-glycine amide (Ac-(gamma-Glu-Cys)n-NH2, n = 2-6) have been synthesized by solid-phase peptide synthesis methods and characterized by 1H NMR spectroscopy. The 1H NMR spectra of the analogue peptides were completely assigned by using band-selective homonuclear-decoupled (BASHD) two-dimensional NMR experiments to achieve spectral resolution. The acid dissociation constant of each cysteine residue in each peptide was determined from chemical shift-pH titration data for the CalphaH protons of the cysteine residues. The resonances for the CalphaH protons were resolved in BASHD-total correlation spectroscopy spectra that were measured as a function of pH. The pKA values for a given thiol group depend on the position of the cysteine residue in the sequence, with the thiol group of the cysteine residue attached to the C-terminal glycine being the most acidic. The pKA values also depend on the size of the peptide, increasing as the size, and thus the negative charge, of the peptide increases. The redox potential for oxidation of the two thiol groups of Ac(gamma-Glu-Cys)2-NH2 to form an intramolecular disulfide bond was also determined by measuring the equilibrium constant for its thiol/disulfide exchange reaction with glutathione.

Characterization of the selenotrisulfide formed by reaction of selenite with end-capped phytochelatin-2.

The phytochelatins are a family of peptides synthesized by plants in response to exposure to heavy metals and metalloids, including selenium in the form of selenite. The amino acid sequence of the phytochelatin (PC) peptides is (gamma-Glu-Cys)n-Gly, where n typically ranges from 2 to 5. In this paper, the products of the reaction of selenite with an end-capped analogue of PC2, Ac-(gamma-Glu-Cys)2-Gly-NH2, are characterized. Selenite reacts with Ac-(gamma-Glu-Cys)2-Gly-NH2 (Ac-PC2-NH2) to form a compound that contains an intramolecular selenotrisulfide (-SSeS-)-linkage (Se[Ac-PC2-NH2]) and oxidized Ac-PC2-NH2. Both Se[Ac-PC2-NH2] and oxidized Ac-PC2-NH2 were isolated by HPLC and were characterized by MALDI-TOF mass spectrometry, by two-dimensional 1H and 13C NMR and, in the case of Se[Ac-PC2-NH2], by 77Se NMR. Using dihedral angles determined from vicinal 1H-1H coupling constants as constraints for the conformations around the Cys(CalphaH)-Cys(CbetaH) bonds, structures were predicted for the most abundant form of both compounds by Monte Carlo molecular mechanics simulations.

Unexpectedly fast cis/trans isomerization of Xaa-Pro peptide bonds in disulfide-constrained cyclic peptides.

Acyclic dithiol and cyclic disulfide forms of the peptides Ac-Cys-Pro-Xaa-Cys-NH2 (Xaa = Phe, His, Tyr, Gly, and Thr) and Ac-Cys-Gly-Pro-Cys-NH2 and the peptide Ac-Ala-Gly-Pro-Ala-NH2 were synthesized and characterized by mass spectrometry and NMR spectroscopy. Rate constants kct and ktc for cis-to-trans and trans-to-cis isomerization, respectively, across the Cys-Pro or Gly-Pro peptide bonds were determined by magnetization transfer NMR techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. It was found that constraints imposed by the disulfide bond confer an unexpected rate enhancement for cis/trans isomerization, ranging from a factor of 2 to 13. It is proposed that the rate enhancements are a result of an intramolecular catalysis mechanism in which the NH proton of the Pro-Xaa peptide bond hydrogen bonds to the proline nitrogen in the transition state. The peptides Ac-Cys-Pro-Xaa-Cys-NH2 and Ac-Cys-Gly-Pro-Cys-NH2 are model compounds for proline-containing active sites of the thioredoxin superfamily of oxidoreductase enzymes; the results suggest that the backbones of the active sites of the oxidized form of these enzymes may have unusual conformational flexibility.