Zinc binding to peptide analogs of the structural zinc site in alcohol dehydrogenase: Implications for an entatic state

Abstract

Zinc binding to the peptide replica and analogs to residues 93-115 of horse liver alcohol dehydrogenase (ADH) was examined by competition of the peptides and the chromophoric chelator 4-(2- pyridylazo)resorcinol for zinc and X-ray absorption fine structure analysis of the zinc ligands. In the enzyme, zinc is coordinated by four Cys residues. In the peptide replica, zinc is bound to three Cys and one His residue. A four-Cys zinc coordination is observed only when His is removed, leading to increased zinc stability. ADH crystal structures reveal that the epsilon-amino group of the conserved residue Lys323 is within H-bond distance of the backbone amide oxygens of residues 103, 105 and 108, likely stabilizing the zinc coordination in the enzyme. The peptide data thus indicate structural strain and increased energy in the zinc-binding site in the protein, characteristic of an entatic state, implying a functional nature for this zinc site.

Medium- and short-chain dehydrogenase/reductase gene and protein families

“…Although the cellular redox potential shifts and is in a constant Table 3. Schematic overview of the peptide set studied [29].…”

“…The results from examination of zinc binding to synthetic peptides corresponding to the structural zinc site of horse liver ADH, with and without amino acid replacements/deletions, indicate that both the number of Cys residues, the presence of His, and the spacing between Cys/His residues are important variables (Table 3) [29]. The metallochromic chelator 4-(2-pyridylazo)resorcinol (PAR) proves to be effective in direct titration of peptide/zinc complexes and allows the determination of zinc binding constants.…”

“…The metallochromic chelator 4-(2-pyridylazo)resorcinol (PAR) proves to be effective in direct titration of peptide/zinc complexes and allows the determination of zinc binding constants. Depending on the number and position of ligands, the zinc binding constants range in an ordered fashion from tight to low binding (from 10 10 to 10 6 M -1 ) [29]. Thus, the binding constants determined by competition with PAR reflect the nature of zinc coordination sites.…”

“…As an alternative, we have examined zinc binding to synthetic peptides corresponding to the structural zinc site of horse liver ADH, with and without amino acid replacements/deletions. The peptide replica of the protein segment (the loop surrounding the structural zinc ion) mimics the metal binding stoichiometry of horse liver ADH [27], and for a peptide set (the 23-residue replica and 17 analogs), zinc stoichiometry, coordination properties and binding affinities were determined and functional interpretations were made [28,29] (Table 3). The use of synthetic peptides to study a zinc binding protein segment and its specific metal coordination properties is an approach with several advantages, including experimental flexibility.…”

“…The use of synthetic peptides to study a zinc binding protein segment and its specific metal coordination properties is an approach with several advantages, including experimental flexibility. In the investigation of the horse liver ADH structural zinc site [29], novel insights regarding zinc ligation and potential reactivity in this protein region were obtained, but it was also realized that a careful examination of available threedimensional structures is vital for interpretations and conclusions. These issues will be discussed in detail after a general background on the multiple roles of zinc in ADHs and metalloproteins in general.…”