Impairment of the catalytic activity of Escherichia coli ribonucleic acid polymerase by a unique reaction of 4-chloro-7-nitrobenzofurazan.
Escherichia coli RNA polymerase loses 55-65% of its catalytic activity on reaction with Nbf-Cl (4-choro-7-nitrobenzofurazan). This partial inactivation was shown to be the result of specific impairment of RNA-chain elongation, since initiation of RNA chains was not altered after treatment with Nbf-Cl. The site of reaction was shown to be a unique thiol on the beta-subunit. This thiol is not accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). No protection of the enzyme against reaction with Nbf-Cl could be obtained with the inhibitor rifamycin nor with calf thymus DNA, GTP or 1,10-phenanthroline, indicating that the unique thiol is probably not within the active site. The specific impairment of RNA-chain elongation thus appears to be the result of a local conformational change which leaves chain initiation unimpaired. Changes observed in the tryptophan fluorescence spectrum of the enzyme or reaction with Nbf-Cl are consistent with formation of a Meisenheimer complex of the reagent with a nucleophilic group on the enzyme near the reactive thiol. It is proposed that formation of such a complex and a subsequent conformational change renders this thiol unusually susceptible to reaction with Nbf-Cl.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010069:Oxadiazoles / Q000494:pharmacology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D010069:Oxadiazoles; D004228:Dithionitrobenzoic Acid; D012321:DNA-Directed RNA Polymerases; D009327:4-Chloro-7-nitrobenzofurazan