Pyruvate Carboxylase Affinity Labelling of the Magnesium Adenosine Triphosphate Binding Site

The 2′,3′‐dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg‐oATP 2− ) was shown to be a linear competitive inhibitor with respect to MgATP 2− in both the acetyl‐CoA‐dependent and ‐independent activities of the enzyme but was a non‐competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg‐oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme. Although bicarbonate, pyruvate and oxaloacetate were ineffective, both MgATP 2− and acetyl‐CoA protected the enzyme against this chemical modification. At 100% inactivation, 1.1 ± 0.1 mol of Mg‐oATP 2− were bound to the enzyme per mole of biotin. Acetyl‐CoA had no effect on this stoichiometry. Chromatography of samples of an enzymic digest of Mg‐o[ 14 C]ATP 2− ‐labelled enzyme revealed one major band of radioactivity which co‐chromatographed with authentic Lys‐oATP.