J. Biol. Chem., Vol. 263, Issue 18, 8635-8641, 06, 1988
pK values for active site residues of carboxypeptidase A
WL Mock and JT Tsay
Department of Chemistry, University of Illinois, Chicago 60680.
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.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
