Effect of metal ions on the activity and stability of wild type and mutant pyrazinamidases

Abstract

Introduction: Pyrazinamidase is a metalloenzyme with hydrolyzing activity which is responsible for conversion of pyrazinamide (anti tuberculosis drug) to active molecule, pyrazinoic acid. The metal-binding site in the enzyme is composed of Asp49, His51, His56 and His71. Mutations in the pyrazinamidase gene are responsible for resistance to pyrazinamide in Mycobacterium tuberculosis and can alter the binding of metal ions to the metal binding site in the enzyme. Therefore, it is important to study the effect of metal ions on the enzymatic activity and stability of the wild type and mutant pyrazinamidases. Materials and methods: In this study, E. coli BL21 was transformed by expression vectors carrying wild type and mutant pyrazinamidase genes. The recombinant proteins were expressed and then purified by Ni- agarose column. The purity of the purified proteins was analyzed by SDS-PAGE electrophoresis. Finally, the activity and stability of the purified enzymes were studied in the presence of 1mM of Ni2+, Fe2+ and Mn2+. Results: SDS-PAGE analysis showed that the expressed enzymes were purified. The activity and stability results depicted that Ni2+ increases the activity and stability of the wild type and mutant (mutant1: L151S, and mutant2 (triple): A143T/T168A/E173K) enzymes. Fe2+ decreased the activity and stability of mutant1, but has no significant effect on other enzymes. The activity and stability of the enzymes decreased in the presence of Mn2+. Discussion and conclusion: Ni2+ interact favorably with metal binding site of the wild type enzyme and mutants compared with Fe2+ and Mn2+ and then increases the activity and stability of the enzymes.

Keywords

• Activity and stability
• Metal binding site
• Mycobacterium tuberculosis
• Pyrazinamidase
• Resistance to pyrazinamide