Catalytic transfer of hydride ions from NADPH to oxygen by the interconversions of proline and Δ 1-pyrroline-5-carboxylate

Abstract

Studies in reconstituted systems indicate that the interconversions of proline and Δ 1-pyrroline-5-carboxylate can constitute a shuttle which transfers reducing equivalents into mitochondria as proline and oxidizing potential out as Δ 1-pyrroline-5-carboxylate. The studies reported here determine if the transfer of reducing equivalents can be stoichiometrically greater than the utilization of shuttle intermediates. First, 3HOH production from [5- 3H]proline was used to quantitate proline oxidation under conditions where Δ 1-pyrroline-5-carboxylate could recycle back to proline in a system containing mitochondrial particles and pyrroline-5-carboxylate reductase. In parallel incubations the metabolic fate of [U- 14C]proline was determined. 3HOH production continued to increase while no net change occurred in 14C-labeled proline and Δ 1-pyrroline-5-carboxylate indicating catalytic activity in this system. In another system that contained intact mitochondria and pyrroline-5-carboxylate reductase, proline oxidation was quantitated by both [U- 14C]proline recoveries and 3HOH production from [5- 3H]proline. Proline oxidation was the same by both methods in incubations lacking NADPH and pyrroline-5-carboxylate reductase. However, it was significantly underestimated by [U- 14C]proline recoveries as compared to 3HOH production in incubations containing all shuttle components. These results indicate catalytic functioning of the proposed shuttle in a reconstituted system containing intact mitochondria. This shuttle may function at specific times to catalytically generate cytosolic NADP + and in turn regulate enzymes limited by [NADP +]. We suggest that the proposed shuttle may function to increase ribose-5phosphate synthesis by the oxidative limb of the pentose phosphate pathway and inturn increase PP-ribose-P and purine synthesis during the initiation of cell growth.