J. Biol. Chem., Vol. 261, Issue 33, 15513-15518, Nov, 1986
On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study
P Gettins
113Cd and 31P NMR have been used to investigate the interactions of
inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as
a replacement for the native zinc atom. In the absence of inhibitor and
over the pH range 6-9, no 113Cd resonance is visible at room temperature.
Upon lowering the temperature to 270 K, however, a broad resonance can be
seen at 120 ppm. These results are discussed in terms of possible sources
for this resonance modulation. Binding of low molecular weight inhibitors
containing potential metal-coordinating moieties results in the appearance
of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a
fact which is reflected in the chemical shift of the cadmium resonance and,
for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P
spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor.
For inhibitors that coordinate to the metal ion via oxygen, the 113Cd
chemical shift is in the range 127-137 ppm, whereas for sulfur coordination
there is a downfield shift of approximately 210 ppm. The complexes of
113Cd- substituted carboxypeptidase A with the D and L isomers of
thiolactic acid are distinguished by a difference of 11 ppm in the chemical
shift of their cadmium resonances. The enzyme complex formed with the
macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites,
shows one or possibly two closely spaced broad 113Cd resonances. Both the
chemical shift and the line width of the 113Cd resonances of the
[113Cd]carboxypeptidase-inhibitor complexes give valuable structural and
dynamic information about the enzyme active site.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?