pK values for active site residues of carboxypeptidase A.
The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.
Mock
W L
WL
Department of Chemistry, University of Illinois, Chicago 60680.
Tsay
J T
JT
D001665:Binding Sites; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy
D007093:Imidazoles; D008024:Ligands; D002268:Carboxypeptidases; D043422:Carboxypeptidases A