Zinc-directed inhibitors for zinc proteinases

Abstract

Zinc proteinases have been recognized as a distinct class of proteolytic enzymes in which at least one ion of zinc is involved directly in catalysis. This family includes a growing number of biologically important enzymes which are attractive targets for rational drug design. In this paper we examine the special features of the zinc binding environment of these enzymes in order to gain information which could be useful in the preparation of 'zinc-directed' selective inhibitors. Carboxypeptidase A (CPA) is presented as a model for one class of zinc proteinases, and the active-site zinc and its interactions are examined with the primary focus on geometrical considerations. The three-dimensional structure of the native and apoenzyme are discussed, together with the high-resolution structure of several enzyme-inhibitor complexes. This paper will first present a structural analysis of CPA derivatives and then discuss a series of zinc model compounds which have been prepared and characterized in order to examine the ligand and geometrical preferences of the zinc in an unstrained system. X-ray crystallography (macromolecular and small molecule) is the main experimental method used for the structural analyses, while complementary computational methods have been used for the examination of electrostatic potentials. The results from the various experimental efforts are assembled in order to draw general conclusions on the potential use of the zinc ion as the primary target for inhibitor binding. The results of these studies suggest that the zinc ion is important for both the binding and the catalytic activation of the substrate as well as for stabilization of the tetrahedral reaction intermediate.

Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.75 Å resolution

“…Figure 13 shows that there is no change in the overall geometry of the active site when the two zinc atoms are removed from the native enzyme, similar to the case of CPA (Feinberg et al, 1993(Feinberg et al, , 1995H.M.G. et al, unpublished results), which contains one zinc cation in the active site.…”

“…In addition to this extra density, the water molecule had refined to a position which put it 2.7 to 2.8 Å from each zinc. This distance is considerably longer than the distance commonly observed for Zn-O interactions in zinc proteins and in small model compounds (Feinberg et al, 1993(Feinberg et al, , 1995, as well as significantly (Ramachandran et al, 1963) for the final structure of SGAP. Glycine residues are shown as triangles; all others as squares.…”

“…(Chevrier et al, 1994) between Asp117 and Asp118, where Asp117 also forms the bridging ligand between the two zinc cations of that structure. As discussed by Chevrier et al, a cis-peptide is not uncommon in, or near the active site of enzymes, and particularly near the ligands of metal ions of metalloenzymes (as for example in carboxypeptidase A (CPA), (Feinberg et al, 1995). The single disulphide bridge in the molecule, involving Cys245 and Cys250, creates a short loop which contains His247, one of the zinc ligands in the active site.…”

“…Replacement of the single zinc ion in CPA with a mercury cation (Rees et al, 1986;H.M.G. et al, unpublished results) produces a partially active enzyme (Coleman & Vallee, 1961) which can bind inhibitors (Feinberg et al, 1993(Feinberg et al, , 1995. The refined structure of mercury replaced SGAP (Figure 14) shows that only one of the two possible zinc sites contains mercury.…”

Thermodynamics of binding of angiotensin-converting enzyme inhibitors to enzyme active site model

“…Zinc proteinases have been recognized as a distinct class of proteolytic enzymes in which at least one ion of zinc is involved directly in catalysis [18]. Bivalent zinc ion is an ideal building part for the hydrolytic enzymes because of its unique electronic configuration: vacant 4s and filled 3d orbital mean redox stability and allow coordination number to vary from 4 to 6, and therefore in the reaction pathway multiple transition states may exist.…”