Gly-His-Thr-Asp-Amide, an Insulin-Activating Peptide from the Human Pancreas Is a Strong Cu(II) but a Weak Zn(II) Chelator

Abstract

The Cu­(II) and Zn­(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV–vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu­(II) chelator, forming a three-nitrogen complex with a conditional affinity constant C K at pH 7.4 of 4.5 × 1012 M–1. The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with C K on the order of several hundred reciprocal molar. The Zn­(II) binding ability of GHTD-am is relatively weak, with C K values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M–1 for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn­(II) and Cu­(II) ions with insulin. A direct effect of GHTD-am on the Zn­(II) interactions with insulin is unlikely, but its Cu­(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.

Ternary Cu(II) Complex with GHK Peptide and Cis-Urocanic Acid as a Potential Physiologically Functional Copper Chelate

“…Such complex corresponds to CuH 2 (GHK) 2 stoichiometry. However, the observed changes in the electronic spectra are very similar to those seen previously in ternary complexes of other Xaa-His peptides (where Xaa is any amino acid residue except Pro) with imidazole (3+1N coordination, where three nitrogen atoms, NH 3 + (N-term) , N amide , and N Im , are from the first peptide molecule, and one nitrogen atom is N Im from the second peptide molecule) [ 20 , 21 ]. Therefore, we assign the 3+1N coordination to this complex and propose to call it “auto-ternary”, with the actual Cu(GHK)(H 2 GHK) stoichiometry.…”

“…Two additional deprotonations occur in the mono -complex at alkaline pH. The first probably corresponds to a water molecule (p K = 9.61) within the equatorial plane of the Cu(GHK) species [ 20 , 21 ]. Another is associated with the ε-amino nitrogen (p K = 10.77) from a side chain of Lys residue.…”

“…Another is associated with the ε-amino nitrogen (p K = 10.77) from a side chain of Lys residue. The aforementioned assignment of the deprotonation to a water molecule instead of an imidazole N1 from another Cu(GHK) complex (with concomitant polynuclear species formation) is rationalized by the lack of drastic changes of CD parameters that would be expected upon formation of imidazole-bridged dimeric or tetrameric species [ 21 , 22 , 23 ].…”

“…Imidazole-based ligands can increase the overall affinity of metal complexes containing tridentate ligands by several orders of magnitude [ 20 , 29 , 30 , 31 , 32 , 33 ]. Urocanic acid, a by-product of L-histidine, contains an imidazole ring that could form a stable ternary Cu(II) complex with GHK in a manner similar to other Xaa-His peptides such as Trp–His–Trp–Ser–Lys–Asn–Arg, Gly–His–Thr–Asp, and Ala–His–His [ 20 , 21 , 34 ].…”

Triggering Cu-coordination change in Cu(ii)-Ala-His-His by external ligands

“…Imidazole is also a relatively strong ternary ligand for XH peptides, as demonstrated recently. 12,19 In these experiments AAH and AH peptides were used as references representing pure 4N and 3N coordination modes, respectively. In the absence of Im, Cu(AHH) shows an absorption band with λmax at 522 nm, in line with the predominant presence (~89%) of the 4N form, (Fig.…”