Inactivation of E. coli RNA polymerase by pyridoxal 5′-phosphate: Identification of a low pKa lysine as the modified residue

The inactivation of E. coli RNA polymerase (3.3 × 10^-7M) by pyridoxal 5′-phosphate (1 × 10^-4M to 5 × 10^-4M) is a first order process with respect to the remaining active enzyme. Studies of the variation of the first order rate constant with the concentration of pyridoxal 5′-phosphate show that the inactivation reaction follows saturation kinetics. The formation of a reversible enzyme-inhibitor intermediate is postulated. Kinetic studies at different pH values indicate that the inactivation rate constant depends on the mole fraction of one conjugate base with pK_a 7.9. The apparent equilibrium constant (association) for the inactivation reaction is independent of the pH and is 1.8 × 10⁴ M^-1. By electrophoretic and chromatographic analysis of enzyme hydrolyzates after pyridoxal 5′-phosphate and NaBH₄ treatment only N-ε{lunate}-pyridoxyllysine was found. It is postulated that a lysine ε{lunate}-amino group with a low pK_a is critical for the activity of the enzyme.