MECHANISTIC STUDIES ON THE INHIBITION OF THERMOLYSIN BY A PEPTIDE HYDROXAMIC ACID

The mechanism of inhibition of thermolysin by the peptide hydroxamic acid HONH-isobutylmalonyl-Ala-GlyNH2 has been probed by 4.rough and temperature dependencies and solvent deuterium isotope effects. We found the following: (1) At pH 6.5 and 25-degrees-C, the K(i) for inhibition of thermolysin by HONH-isobutylmalonyl-Ala-GlyNH2 is 63 +/- 5 nM and reflects a potency for this compound not previously appreciated. (2) The Ph dependence of 1/K(i) at 25-degrees-C is bell-shaped with pK(al) = 5.4 +/- 0.1, pK(a2) = 8.2 +/- 0.1, and (K(i)limit = 56 +/- 4 nM. These pK(a) values are similar to those that we obtained from the pH dependence of k(c)/K(m) for the thermolysin-catalyzed hydrolysis of 3-(2-furyl)acryloyl-Gly-Leu-Ala (hydrolysis at Gly-Leu) and suggest that the active site amino acid residues that are involved in catalysis are also involved in binding this inhibitor. The pH dependence of 1/K(i) also indicates that thermolysin binds the inhibitor as the neutral, un-ionized acid and not as an anion, as suggested previously by other workers [Holmes, M. A.; Matthews, B. W. Biochemistry 1981, 20, 6912. Nishino, N.; Powers, J. C. Biochemistry 1978, 17, 2846. Nishino, N.; Powers, J. C. Biochemistry 1979, 18, 4340]. (3) At pH 6.5, values of K(i) increased with increasing temperature from 18 nM at 5-degrees-C to a plateau of 200 nM between 45 and 60-degrees-C. The van't Hoff plot of this data was analyzed according to a two-step model involving the formation of an initial complex, (E:I)1, that undergoes a conformational isomerization to a second complex, (E:I)2, at high temperature. At temperatures less than 35-degrees-C, only (E:I)1 accumulates and, thus, entirely accounts for inhibition at temperature less than 35-degrees-C. (4) The solvent deuterium isotope effect on K(ass) (= K(ass,H2O)/K(ass,D2O), where K(ass) = 1/K(i)) is 0.74 +/- 0.02 and, like solvent isotope effects for TLN catalysis [D2O(k(c)/K(m)) = 0.74; Izquierdo, M.; Stein, R. L. J. Am. Chem. Soc. 1990, 112, 6054], originates from the transfer of a zinc-bound water molecule to bulk solvent. Based on these results, a mechanism for the inhibition of thermolysin is formulated and discussed.