Mechanistic implications of cyanide binding to carboxypeptidase B.
The putative metal coordinating ligand cyanide was used to study the effects of modifications of the metal coordination sphere on the spectral properties and catalytic activity of cobalt and zinc carboxypeptidases. The absorption spectra of Co2+-carboxypeptidase B in the presence of cyanide pointed to a direct interaction of the ligands with the metal. Gel-filtration experiments showed that the binding of one mole of ligand per mole of enzyme metal ion resulted in maximal spectral effects. Binding of cyanide to the metal ion as measured by absorption spectroscopy was inhibited by acetyl-L-arginine, a peptide pseudosubstrate, and by acetyl-D-arginine, a competitive peptide inhibitor. Addition of acetyl arginine to the enzyme-cyanide complex caused displacement of the ligand, as evidenced by the spectral parameters. Cyanide inhibited peptide hydrolysis in a partially noncompetitive manner, i.e. it did not prevent binding of the substrate to the enzyme but the enzyme-substrate-cyanide complex was hydrolyzed at a slower rate than the enzyme-substrate complex. The dissociation constant evaluated from kinetic studies for the binding of cyanide to Co2+-carboxypeptidase B was in good agreement with that obtained from spectral measurements. Hydrolysis of the ester analog of the basis peptide substrate was not affected by cyanide. Based on these data a model is proposed in which the peptide carboxyl group displaces the water molecule from the metal coordination sphere during catalysis without increasing the coordination number.
D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism*; D003035:Cobalt / Q000032:analysis; D003486:Cyanides / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D011485:Protein Binding; D013552:Swine; D015032:Zinc / Q000494:pharmacology
D003486:Cyanides; D008024:Ligands; D003035:Cobalt; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D015032:Zinc