Structure and mechanism of Mycobacterium smegmatis polynucleotide phosphorylase

Effect of alanine mutations of enzymic metal ligands. Reaction mixtures (70 µL) containing 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 0.5 mM (NH₄)₂PO₄, 0.1 µM 5′ ³²P-labeled 24-mer RNA, and 0.1 µM wild-type or mutant PNPase homotrimer as specified above the gels were incubated at 37°C. Aliquots (10 µL) were removed at the times specified above the lanes and quenched immediately with formamide/EDTA. The reaction products were analyzed by urea-PAGE and visualized by autoradiography.