Magnetic resonance investigation of ionizable residues at the active site of thermolysin.
The details of the pH dependence of the thermodynamic and magnetic interactions of the active-site region of thermolysin in which manganese has replaced the active-site zinc atom and the inhibitor N-trifluoroacetyl-D-phenylalanine have been examined. These show a number of ionizable groups in the active-site region. A cooperative displacement of manganese at the catalytic site is observed as pH is lowered. This appears to be the result of the protonation of histidine-142 and -146 which act as metal ligands. The metal is 50% displaced at pH 6.0. At higher pH values, the environment of the bound manganese changes as a result of the ionization of at least two groups of approximate pKa = 8.5 and 9.5. These values are assigned to tyrosine-157 and to the water molecule which acts as a metal ligand at the active site. The binding behavior of the inhibitor strongly suggests that two molecules of inhibitor bind to the enzyme. The weaker site is competitive with the synthetic substrate FAGLA (furylacryloylglycyl-leucinamide), while the strong site has no effect on FAGLA hydrolysis. This second site is in the vicinity of the active site with a distance of 8 A or less between the trifluoromethyl group and manganese bound at the active site.
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese; D008433:Mathematics; D010649:Phenylalanine / Q000031:analogs & derivatives; D011487:Protein Conformation; D013820:Thermolysin
D008345:Manganese; D010649:Phenylalanine; D013820:Thermolysin