title: A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion.
abstract: The 13C-n.m.r. titration shifts of the alpha-methylene group of N-alkylated imidazoles are shown to be a sensitive probe of the ionization of the imidazolium ion. The 13C-n.m.r. titration shifts of both the intact and denatured/autolysed 2-13C- and 1-13C-enriched trypsin-7-amino-3-benzyloxycarbonylamino-1-chloroheptan-2-one (Z-Lys-CH2Cl) complexes are compared. The titration shift for the denatured/autolysed complex confirms that this ionization is due to deprotonation of the N-alkylated imidazolium ring of histidine-57. In the intact trypsin-inhibitor complex the titration shift due to the 1-13C-enriched carbon is anomalous. We conclude that this titration shift cannot arise solely from the ionization of the imidazolium ion of histidine-57 and that the pKa of the imidazolium ion of histidine-57 is raised in the trypsin-inhibitor complex. The relevance of these studies to the mechanism of action of the serine proteinases is discussed.
mesh_terms: D000478:Alkylation; D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D011489:Protein Denaturation; D014361:Trypsin Inhibitors / Q000378:metabolism*
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D007477:Ions; D046911:Macromolecular Substances; D014361:Trypsin Inhibitors; D006639:Histidine; C047455:7-amino-3-benzyloxycarbonylamino-1-chloroheptan-2-one