The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation.
1. By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 X 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.
The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation. 1. By using Bio-Gel A1.5M and Sephadex G-150 columns, crude cell-free extracts of Escherichia coli were fractionated to demonstrate the existence of a proline-biosynthetic aggregate. 2. Sephadex G-150 resolves two glutamyl kinases that are inhibited by proline, with mol.wts. of 125000 and 38000, the reactions of which are Mg2+-dependent. The heavier species is more sensitive to inhibition by proline. 3. Gamma-Glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase (EC 1.5.1.2) have mol.wts. of approx. 125000 and 190000 respectively, the specific activity of the latter being 5 X 10(3)-fold greater than either of the other two biosynthetic enzymes or of the total pathway in vivo. 4. Bio-Gel A1.5M chromatography gave a single glutamyl kinase of mol.wt. 250000, and the possibility of this being a constituent of an enzyme complex is discussed.