Abstract
The basicity of His 57-Nepsilon2 within the low-barrier hydrogen-bonded (LBHB) diad His 57-Asp 102 and the 1H NMR chemical shift of the LBHB proton in tetrahedral, hemiketal complexes of chymotrypsin with peptidyl trifluoromethyl ketones (peptidyl-TFKs) have been studied. The following results were obtained with various peptidyl-TFKs at 5 degrees C: N-Ac-Gly-DL-Phe-CF3, pKa = 11.1 and deltaLBHB = 18.7 ppm; N-Ac-L-Val-DL-Phe-CF3, pKa = 11.8 and deltaLBHB = 18.9 ppm; N-Ac-L-Leu-DL-Val-CF3, pKa = 10.3 and deltaLBHB = 18.9 ppm; and N-Ac-L-Leu-DL-naphthyl-CF3, pKa = 10.9 and deltaLBHB = 19.0 ppm. Results for peptidyl-TFKs with Phe in the P1 position and N-Ac, N-Ac-Gly, N-Ac-L-Val, and N-Ac-L-Leu in the P2 position were well correlated with literature values for inhibition constants Ki and kcat/Km for the corresponding peptidyl methyl esters. The plot of log Ki versus the apparent pKa of His 57-Nepsilon2 displayed a slope of -0.77, and that of log kcat/Km for peptidyl methyl esters versus the pKa of His 57-Nepsilon2 in corresponding peptidyl-TFK complexes gave a slope of 0.68. The slope of a plot of pKa versus deltaLBHB was 3.7, and that of log kcat/Km for peptidyl methyl ester substrates versus deltaLBHB for the corresponding peptidyl-TFK-chymotrypsin complexes was 2.7. A plot of log Ki versus deltaLBHB displayed a slope of -3.0. These plots indicated that the pKa of His 57 and substrate reactivity were correlated with increasing strength of the low-barrier hydrogen bond. The apparent pKa of His 57-Nepsilon2 for the chymotrypsin-N-Ac-L-Leu-DL-Phe-CF3 complex is 10.6 at 25 degrees C, whereas it is 12.0 at 5 degrees C [Cassidy, C. S., Lin, J. L., and Frey, P. A. (1997) Biochemistry 36, 4576-4584]. The apparent discrepancy is likely to be due to a temperature dependence in the cooperative ionization of His 57 in peptidyl-TFK complexes, which appears to be coupled to inhibitor dissociation, hydration and ionization of free peptidyl-TFK, ionization of Ile 16, and a conformational change.
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