Grobelny D, Goli U B, Galardy R E
Department of Biochemistry, University of Kentucky, Lexington 40536.
Biochemistry. 1989 Jun 13;28(12):4948-51. doi: 10.1021/bi00438a006.
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)
