BOC-GLU-THR-ILE-HIS-OME ZN2+ IN TERMS OF THE ZINC-BINDING SITES OF PROTEASES/

Reaction of Boc-Glu-Thr-Ile-His-OMe (Boc: butoxy carbonyl) (H(2)1), an d Zn(ClO4)(2). 6H(2)O in N,N-dimethylformamide (DMF) was studied using NMR and mass spectrometry with respect to the active sites of proteas es. The study of H(2)1/Zn2+ = 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 Glu-carboxyl group still remains in the form of -COOH. Mass spectrometry showed that Zn2+ in H (2)1/Zn2+ attracts water molecules under the ionization conditions of fast atom bombardment. The temperature (T) dependence of H-1 NMR of H( 2)1/Zn2+ = 1:1 in DMF-d(7), studied in the temperature range 218-323 K , revealed that the Zn2+ unit undergoes protonation and hydration at l ow temperatures (below 253 K). The chemical shifts (S) of the amide pr otons were nevertheless linearly dependent on temperature over the ran ge 218-323 K, meaning that the 1:1 solution obeys the common reaction equilibrium and that the averaged main-chain folding of the peptide mo iety is conserved. Based on this finding, the distance geometry and re strained molecular dynamics analyses were carried out using the distan ce information on 61 H-1-H-1 pairs, extracted from the rotating frame nuclear Overhauser effect spectroscopy experiment on this solution (30 0 K). These analyses revealed that the averaged main-chain folding of the Glu-Thr-ne-His is just superimposed on that of the Zn2+ binding si tes of proteases forming a single helix with the Glu carboxyl group si tuated in the vicinity of the His imidazole group, or is directly but distally bound to Zn2+ (in the case of gamma-COOGlu-). The hydrogen bo nd between gamma-COOGlu- and the H2O on Zn2+ was also found in compara tively higher energy area. Gel chromatography of the dried material ob tained from H(2)1/Zn2+ = 1:1 mixture gave only [(H1)(2)Zn] (2), as a d iscrete compound. The coordination atoms to Zn2+ in 2 were assigned as Glu amide (N-) and His imidazole nitrogens. (C) 1998 Elsevier Science S.A. All rights reserved.