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 pKa 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-pyridoxyllysine was found. It is postulated that a lysine ε-amino group with a low pKa is critical for the activity of the enzyme.