Binding energetics of phosphorus-containing inhibitors of thermolysin.
The importance of a specific hydrogen bond between thermolysin and a phosphonamidate inhibitor, Z-NHCH2-PO(O-)-Leu-Leu (1) [Bartlett, P. A., & 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 phosphonamidate 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----3 and 4----6) weakens the overall binding free energy by about 1.5 kcal/mol. A negligible 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., & 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----2 [Bartlett, P. A., & Marlowe, C. K. (1987) Science (Washington, D.C.) 235, 569-571] and 4----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 5 (and by inference between 1 and 2) is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, University of Kentucky, Lexington 40536.
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Grobelny
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Goli
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Galardy
D001665:Binding Sites; D004735:Energy Transfer; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D012995:Solubility; D013816:Thermodynamics; D013820:Thermolysin / Q000037:antagonists & inhibitors*
D008024:Ligands; D009943:Organophosphorus Compounds; D013820:Thermolysin