Nuclear Magnetic Resonance Studies of α-Chymotrypsin. I. NMR Studies of the Binding of Small Molecule Inhibitors to α-Chymotrypsin. II. NMR Studies of the Interaction of N-TFA-D-Tryptophan Semicarbazide with α-Chymotrypsin. III. ¹³C-NMR Studies of Methylated α-Chymotrypsin. IV. NMR Studies of Acylated Chymotrypsins

Part I Magnetic resonance studies of the interaction of N-trifluoro-acetyl-D-(and L-)p-fluorophenylalanine and N-trifluoro-acetyl-D-(and L-)tryptophan with α-chymotrypsin have been carried out at pH 5.0-8.0. The effect of enzyme oligomerization and competitive inhibition have been quantitatively accounted for. The trifluoro- acetyl group of the D-isomer of both inhibitors is directed toward the active site of the enzyme, while that of the L-isomers is directed toward Ser 214. The aromatic side chain of all inhibitors resides in the hydrophobic specificity pocket of the enzyme. Ionization of a group on the free enzyme with pKa of 6.6 (presumably His 57) leads to a sharp decrease in binding affinity of the enzyme for anionic inhibitor molecules. Part II Nuclear magnetic resonance studies of the binding of N-trifluoroacetyl-D-tryptophan semicarbazide at pH 5.0-9.0 have been carried out. Ionization of a group on the free enzyme (tenatively assigned to His 40) causes a nine-fold increase in the enzyme-inhibitor dissociation constant. Neutralization of His 57 affects only the chemical shift of the bound inhibitor molecule. Part III Carbon -13 nuclear magnetic resonance studies of methylated α-chymotrypsin have been carried out. Selective enrichment of the modifying methyl group allows assignment of this resonance. The pH-dependent shift of the methyl group has been measured and indicates a pKa of 6.75. The direction of the shift upon ionization (30 Hz downfield) indicates that this ionization is accompanied by a conformational change.