The reactive lysine residue at the allosteric site of sheep kidney pyruvate carboxylase

Investigations have revealed that sheep kidney pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) possesses a lysine residue whose integrity is essential for enzymic activity. Modification of the enzyme with amino group reagents such as 1-fluoro-2,4-dinitrobenzene, trinitrobenzene sulphonic acid and potassium cyanate resulted in a loss of catalytic activity. The characterisation of the major radioactive dinitrophenyl-amino acid derivative of 1-fluoro-2,4-dinitro[14C]benzene labelled enzyme as e-dinitrophenyl-lysine confirmed the identity of the reactive residue. Inhibition of the enzyme by 1-fluoro-2,4-dinitrobenzene and trinitrobenzene sulphonic acid was first order with respect to time and inhibitor concentration. From the kinetic data obtained, it was concluded that an average of 1 molecule of these reagents reacts with the enzyme causing inactivation. The rate of inactivation with these reagents was pH dependent, increasing with increasing pH values. The ability of the allosteric effector, acetyl-CoA, to protect the enzyme against inhibition by these reagents suggested that the e-amino group of lysine may be involved in the enzyme-acetyl-CoA interaction. The dissociation constant for the enzyme-acetyl-CoA complex was 5.4·10−4 M.