THE NATURE OF POTASSIUM FERRATE INACTIVATION OF PORCINE MUSCLE ADENYLATE KINASE AND OF OTHER PHOSPHATE BINDING ENZYMES (ACTIVE-SITE-SPECIFIC)

MARY DOLORES CRIVELLONE, Purdue University

Abstract

Potassium ferrate is an analog of phosphate ion and a potent oxidizing agent. The ferrate ion is similar in geometry and electrostatic properties to orthophosphate. Previous studies have demonstrated that ferrate inactivates enzymes in a phosphate-site-specific manner, causing a selective oxidation of key amino acids located in the phosphate binding regions of these enzymes. An examination of the phosphate binding sites of adenylate kinase was undertaken using potassium ferrate. This reagent was found to cause irreversible inactivation of the enzyme. Inhibition could be prevented by the presence of competitive inhibitors or substrates, establishing that the action of ferrate was site-specific. Amino acid and peptide sequence analysis showed that ferrate treatment resulted in the loss of Cys-25 and Tyr-95. Tentative evidence suggests that Tyr-95 is essential for enzyme activity. The involvement of these residues at the phosphate binding sites of adenylate kinase is discussed. The value of potassium ferrate as a general inhibitor of enzymes which recognize compounds possessing phosphoryl groups was investigated. Various types of enzymes which interact with phosphate-containing substances were found to be inactivated by ferrate. Protection by specific substrates and competitive inhibitors indicated that ferrate inactivation was site-specific.

Degree

Ph.D.

Subject Area

Biochemistry

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