title: pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.
abstract: 1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Center for Biochemical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: D S
authors.initials: DS
authors.lastname: Auld
authors.forename: I
authors.initials: I
authors.lastname: Bertini
authors.forename: A
authors.initials: A
authors.lastname: Donaire
authors.forename: L
authors.initials: L
authors.lastname: Messori
authors.forename: J M
authors.initials: JM
authors.lastname: Moratal
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043422:Carboxypeptidases A; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy
chemical_list: D007477:Ions; D002268:Carboxypeptidases; D043422:Carboxypeptidases A