Binding energetics of phosphorus-containing inhibitors of thermolysin

Abstract

The importance of a specific hydrogen bond between thermolysin and a phosphoamidate inhibitor, Z-NHCH2-PO(O-)-Leu-Leu (1) [Bartlett, P.A., and Marlowe, C. K. (1987) Science (Washington, D.C.) 235, 569-571], has been reevaluated. We have determined the inhibition constants (binding free energies) for thermolysin of phosphonamide n-hexyl-P(O)(O-)-Leu-Trp-NHMe (4), phosphonate n-hexyl-P-(O)(O-)OCH(iBu)CO-Trp-NHMe (5), and phosphinates n-hexyl-P(O)(O-)CH2CH(iBu)CO-Trp-NHMe (6) and Z-NHCH2PO(O-)CH2CH(iBu)CO-Leu (3). Replacement of the P-NH group by P-CH2 (1 .fwdarw. 3 and 4 .fwdarw. 6) weakens the overall binding free energy by about 1.5 kcal/mol. A negligile difference in solvation energy has been measured for these pairs, and the basicity of the P-O- ligand for zinc in each pair remains nearly unchanged as determined by pH titration of their 31P NMR resonances. Therefore, this value of 1.5 kcal/mol can be assigned to the specific hydrogen bond known to exist between the P-NH of 1 and thermolysin [Tronrud, D. E., Holden, H. M., and Matthews, B.W. (1987) Science (Washington, D.C.) 235, 871-574] and inferred to exist between 4 and the enzyme. Substitution of P-O for P-NH(1 .fwdarw. 2 [Bartlett, P. A., .fwdarw. Marlowe, C. K. (1987) Science (Washington, D.C.) 235, 569-571] and 4 .fwdarw. 5) weakens the overall binding free energy by 4.1 kcal/mol for each pair as the basicity of the P-O- ligand decreases by about 1.6 pH units. The measured solvation energy difference between 4 and 6 (and by inference between 1 and 2) is negligible. Therefore, we postulate that the loss of 4.1 kcal/mol in binding energy for this substitution results from the loss of a specific hydrogen bond of energy 1.5 kcal/mol present in both phosphonamidates 1 and 4 and a loss of 2.6 kcal/mol in the energy of liganding of the less basic phosphonate inhibitors 2 and 5.