CORRELATIONS OF THE BASICITY OF HIS-57 WITH TRANSITION-STATE ANALOG BINDING, SUBSTRATE REACTIVITY, AND THE STRENGTH OF THE LOW-BARRIER HYDROGEN-BOND IN CHYMOTRYPSIN

The basicity of His 57-N-epsilon 2 within the low-barrier hydrogen-bon ded (LBHB) diad His 57-Asp 102 and the H-1 NMR chemical shift of the L BHB proton in tetrahedral, hemiketal complexes of chymotrypsin with pe ptidyl trifluoromethyl ketones (peptidyl-TFKs) have been studied. The following results were obtained with various peptidyl-TFKs at 5 degree s C, N-Ac-Gly-DL-Phe-CF3, pK(a) = 11.1 and delta(LBHB) = 18.7 ppm; N-A c-L-Val-DL-Phe-CF3, pK(a) = 11.8 and delta(LBHB) = 18.9 ppm; N-Ac-L-Le u-DL-Val-CF3, pK(a) = 10.3 and delta(LBHB) = 18.9 ppm; and N-Ac-L-Leu- DL-naphthyl-CF3, pK(a) = 10.9 and delta(LBHB) = 19.0 ppm Results for p eptidyl-TFKs with Phe in the P-1 position and N-Ac, N-Ac-Gly, N-Ac-L-V al, and N-Ac-L-Leu in the P-2 position were well correlated with liter ature values for inhibition constants K-i and k(cat)/K-m for the corre sponding peptidyl methyl esters. The plot of log K-i versus the appare nt pK(a) of His 57-N-epsilon 2 displayed a slope of -0.77, and that of log k(cat)/K-m for peptidyl methyl esters versus the pK(a) of His 57- N-epsilon 2 in corresponding peptidyl-TFK complexes gave a slope of 0. 68, The slope of a plot of pK(a) versus delta(LBHB) was 3.7, and that of log k(cat)/K-m for peptidyl methyl ester substrates versus delta(LB HB) for the corresponding peptidyl-TFK-chymotrypsin complexes was 2.7, A plot of log K-i versus delta(LBHB) displayed a slope of -3.0, These plots indicated that the pK(a) of His 57 and substrate reactivity wer e correlated with increasing strength of the low-barrier hydrogen bond . The apparent pK(a) of His 57-N-epsilon 2 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) Bioche mistry 34, 4576-4584], The apparent discrepancy is likely to be due to a temperature dependence in the cooperative ionization of His 57 in p eptidyl-TFK complexes, which appears to be coupled to inhibitor dissoc iation, hydration and ionization of free peptidyl-TFK, ionization of I le 16, and a conformational change.