Active site-directed inhibition of E. coli DNA-dependent RNA polymerase by pyridoxal 5′-phosphate

DNA-dependent RNA polymerase from E. coli is rapidly inhibited by preincubation with pyridoxal 5′-phosphate. The enzyme is not inhibited by pyridoxamine 5′-phosphate or pyridoxine. Pyridoxal is about 10 times less effective than pyridoxal 5′-phosphate. Nucleoside triphosphates but not DNA, protect the enzyme from inhibition. Spectral data from the reaction mixture and NaBH4 reduction products indicate the formation of a Schiff base between the aldehyde group of pyridoxal 5′-phosphate and amino group of the enzyme. The results show that the inhibitor is reacting with a critical amino group presumably at the nucleoside phosphate binding site of the active center of the enzyme.