The Three Dimensional Structure of NADP ⁺ Specific Isocitrate Dehydrogenase of Escherichia Coli

Abstract

The NADP + specific isocitrate dehydrogenase (IDH) three-D s - isocitrate: NADP + : oxidoreductase (decarboxylating), E.C. 1.1.1.42. of Escherichia coli was recently purified to electrophoretic homogeneity. The enzyme has a molecular weight of 83,000 daltons, exists as a catalytically active dimer with identical subunits of a molecular weight of 42,000 daltons and an isoelectric point of pH 5. The native enzyme crystallizes in the form of a regular octahedron ranging in size from 5 to 30 um. In this investigation the subunit structure (tertiary Structure) and quaternary structure of E. coli IDH is described along with functional implications. Electrophoretically pure E. coli (F 143/KL259) IDH (.43 mg/ml, specific activity 30.5 enzyme units/mg protein) was maintained in 0.0125 M citrate-phosphate (pH 5.5) containing 20% (v/v) glycerol and 2mM MgCl 2 at 4°C. Fenestrated 400 mesh copper grids were pre-coated with gold before overlaying a thin carbon film (∼30Å thick). Enzyme was deposited on the support film by lightly touching a drop of enzyme-buffer solution, followed by positive staining with 1% uranyl acetate (pH 4.7), rinsed gently in the citrate-phosphate buffer and air dried. Denaturation of IDH enzyme complex into its constituent subunits was effected by treating grids containing enzyme with 10M urea and stained and rinsed as indicated above.