Boc-Glu-Thr-Ile-His-OMe/Zn 2+ in terms of the zinc-binding sites of proteases

Abstract

Reaction of Boc-Glu-Thr-Ile-His-OMe (Boc: butoxy carbonyl) (H 2 1), and Zn(ClO 4) 2·6H 2O in N,N-dimethylformamide (DMF) was studied using NMR and mass spectrometry with respect to the active sites of proteases. The study of H 21/Zn 2+ = 1:1 mixture in DMF-d 7 revealed that Zn 2+ is bound to the His-imidazolyl group. The results of IR experiments on the 1:1 dried material suggested that the Glucarboxyl group still remains in the form of -COOH. Mass spectrometry showed that Zn 2+ in H 2 1/Zn 2+ attracts water molecules under the ionization conditions of fast atom bombardment. The temperature ( T) dependence of 1H NMR of H 2 1/Zn 2+ = 1:1 in DMF-d 7, studied in the temperature range 218–323 K, revealed that the Zn 2+ unit undergoes protonation and hydration at low temperatures (below 253 K). The chemical shifts ( δ) of the amide protons were nevertheless linearly dependent on temperature over the range 218–323 K, meaning that the 1:1 solution obeys the common reaction equilibrium and that the averaged main-chain folding of the peptide moiety is conserved. Based on this finding, the distance geometry and restrained molecular dynamics analyses were carried out using the distance information on 61 1H— 1H pairs, extracted from the rotating frame nuclear Overhauser effect spectroscopy experiment on this solution (300 K). These analyses revealed that the averaged main-chain folding of the Glu-Thr-Ile-His is just superimposed on that of the Zn 2+ binding sites of proteases forming a single helix with the Glu carboxyl group situated in the vicinity of the His imidazole group, or is directly but distally bound to Zn 2+ (in the case of γ-COO Glu −). The hydrogen bond between γ-COO Glu − and the H 2O on Zn 2+ was also found in comparatively higher energy area. Gel chromatography of the dried material obtained from H 2 1/Zn 2+ = 1:1 mixture gave only [(H 1) 2Zn] ( 2), as a discrete compound. The coordination atoms to Zn 2+ in 2 were assigned as Glu amide (N −) and His imidazole nitrogens.