Pyruvate carboxylase: affinity labelling of the pyruvate binding site.

The active-site-directed reagent, bromopyruvate has been used to covalently label the pyruvate binding site of pyruvate carboxylase (E.C.6.4.1.1.) isolated from sheep liver. Oxalo-acetate proved to be the most effective reaction component in protecting the enzyme against inactivation; pyruvate was less effective although its efficiency was enhanced by the presence of acetyl CoA. The other reaction components, MgATP^2? and HCO₃^? failed to protect the enzyme against inactivation. Using bromo[2¹⁴C]pyruvate, it was shown that at 100% inactivation, 1.5 pyruvyl residues were bound per mole of biotin and when the reaction was carried out in the presence of acetyl CoA, this ratio was reduced to 1.0. Analysis of pronase digests of the enzyme revealed that more than 90% of the radioactivity was present as carboxy-hydroxyethyl cysteine.