Ornithine Cyclase (Deaminating)

A protein that converts l-ornithine to l-proline and ammonia has been partially purified (up to 37-fold) from Clostridium PA 3679. The preparation contained a very active Δ1-pyrroline-5-carboxylate reductase (l-proline:NAD(P) 5-oxidoreductase, EC 1.5.1.2), but this enzyme is not involved in the reaction. The cyclase was completely separated from the reductase activity by filtration through Sephadex G-200, centrifugation in a linear sucrose gradient, and polyacrylamide gel electrophoresis. The cyclase activity in the peak fractions from these treatments was very unstable. Since the protein with cyclase activity has a much smaller sedimentation coefficient than the Δ1-pyrroline-5-carboxylate reductase (5.7 versus 10.7), it could not be an aggregate containing the reductase. The partially purified cyclase had a pH optimum of 8.0 to 8.2 and a temperature optimum of 44°. NAD+ was required for a maximum activity but some response was evident when high levels of NADP+ or FAD+ were added. Lineweaver-Burk plots for NAD+ and NADP+ were linear and the apparent Km values were 6.1 x 10-3 and 3.3 mm, respectively. The enzyme is specific for l-ornithine and the apparent Km is 11.1 mm. The products are l-proline and ammonia. No tritium was incorporated into either ornithine or proline when the cyclase reaction was carried out in 3H2O.