13C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A.
Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.
Bertini
I
I
Department of Chemistry, University of Florence, Italy.
Monnanni
R
R
Pellacani
G C
GC
Sola
M
M
Vallee
B L
BL
Auld
D S
DS
D000085:Acetates / Q000494:pharmacology*; D002247:Carbon Isotopes; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010648:Phenylacetates / Q000494:pharmacology*; D011485:Protein Binding
D000085:Acetates; D002247:Carbon Isotopes; D010648:Phenylacetates; D003035:Cobalt; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; C025136:phenylacetic acid