Untitled

Abstract

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, JBiol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution.The structure of the Co2+-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobaltrelative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd2+-and Mn2+-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 1 4 4 . Some of these sidechain movements are similar to adjustments that have been observed previously in association with the "hingebending" motion that is presumed to occur during catalysis by the zinc endoproteases.In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect. (Fig. 1) showed the zinc liganded tetrahedrally by His 142, His 146, Glu 166, and a water molecule, hereafter referred to as Wat 231 (Fig. 2). Further refinement has suggested that, in addition to Wat 23 1, there is also a dipeptide, presumably a product of selfproteolysis, bound to the active site (Holland et al., 1992 (1974, 1976) have shown that removal of zinc from thermolysin yields an inactive apoenzyme and that varying levels of esterase and peptidase activity can be restored upon substitution of the native zinc atom with transition metals. In particular, Zn2+, Co2+, and Mn2+, when added in stoichiometric amounts, restored 100,200, and 10% of the activity of the native enzyme toward furylacyloylglycyl-L-leucyl amide (FAGLA). Furthermore, in concentrations up to 1 mM, these metals retained or restored catalytic activity to some extent. Fe2+ in high molar excess restored about 60% of native activity. Zn2+ in excess of the amount required for catalytic activity actually inhibited the enzyme. The transition metals Mg2+, Cr2+, Ni2+, Cu2+, Mo2+, Pb2+, Hg2+, Cd2+, Nd2+...