Identification of histidine-119 as the target in the site-specific inactivation of ribonuclease A by ferrate ion.
Ferrate ion, a powerful oxidant which is an analog of orthophosphate ion, has previously shown some promise as a site-specific probe of enzymes which interact with phosphate compounds. In order to explore the general applicability of this reagent, it has been tested against ribonuclease A, an enzyme whose structure and active center have been well described. Treatment with a molar ratio of ferrate to enzyme of less than 20 leads to a loss of 87% of the activity. The known competitive inhibitors, 2'-cytidylic acid, inorganic pyrophosphate, and orthophosphate all protect the enzyme from inactivation. Inactivation is accompanied by a loss of the capacity to bind 2'-cytidylic acid. Ferrate inactivation at pH 5.0 is accompanied by the modification of only one amino acid. The amino acid which was identified by amino acid and sequence analyses of peptide fragments obtained by cyanogen bromide treatment and selective proteolysis proved to be histidine-119, whose essential role at the active center has long been established.