Chemical modification of Escherichia coli RNA polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity and intrinsic zinc binding.
RNA polymerase (RPase) from Escherichia coli contains five subunits (alpha 2 beta beta' sigma) and two intrinsic Zn ions located in the beta and beta' subunits. This enzyme was rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The difference spectrum of the DEP-inactivated and native RPases showed a single peak at 240 nm indicating the formation of N-carbethoxyhistidines. No decrease in absorbance at 278 nm, due to O-carbethoxytyrosine, or modification of amino and sulfhydryl groups was observed. Inactivated RPase with six to nine histidines being modified could be fully reactivated by incubation with 0.5 M hydroxylamine at pH 6.0 and room temperature for 1 h. No structural difference was detected between the native and modified enzymes as evidenced by UV/visible and fluorescence spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern, or gel filtration properties. Substrate ATP at 0.11 and 1.14 mM concentrations provided, respectively, 25% and 90% protection against DEP inactivation, while template DNA did not. These results suggest that one or more histidine residues is/are in close proximity to the substrate binding site. The pH dependence of the DEP inactivation of RPase suggested the modification of histidine at the active site with a pK value of 6.9. The inactivation of RPase by DEP and the formation of N-carbethoxyhistidine displayed a similar second-order rate constant of approximately 0.9 mM-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D013053:Spectrophotometry; D015032:Zinc / Q000378:metabolism*
D005561:Formates; D006898:Hydroxylamines; D046911:Macromolecular Substances; D019811:Hydroxylamine; D006639:Histidine; D012321:DNA-Directed RNA Polymerases; D015032:Zinc; D004047:Diethyl Pyrocarbonate