Correlations of the basicity of His 57 with transition state analogue binding, substrate reactivity, and the strength of the low-barrier hydrogen bond in chymotrypsin.
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.
Institute for Enzyme Research, The Graduate School, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53705, USA.
J
J
Lin
C S
CS
Cassidy
P A
PA
Frey
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry; D004789:Enzyme Activation; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D004151:Dipeptides; D007659:Ketones; D046911:Macromolecular Substances; C054856:N-acetyl-leucyl-phenylalanyl trifluoromethyl ketone; D006639:Histidine; D001216:Asparagine; D002918:Chymotrypsin