The Metabolism of Proline in Higher Plants: II. L-PROLINE DEHYDROGENASE FROM COTYLEDONS OF GERMINATING PEANUT (ARACHIS HYPOGAEA L.) SEEDLINGS

The L-proline-dependent reduction of NAD⁺ has been obtainedwith a soluble enzyme extracted from acetone powders of thecotyledons of 3- to 5-day-old germinating peanut seedlings.The enzyme has been purified approximately 20-fold. NAD⁺ ismuch more effective as an electron acceptor than NADP⁺, thereaction rate with the latter being only 15 per cent that withthe former. The K_m for L-proline at pH 10.3, with NAD⁺ saturating,is 0.30 mM, and that for NAD⁺, with L-proline saturating, is0.25 mM. NADP⁺ is an excellent competitive inhibitor for NAD⁺with a K₁ of 6.2 µM. L-proline, L-proline methyl ester, and 3,4-dehydro-DL-prolineare equally effective as substrates. Thiazolidine-4-carboxylatecatalyses the reduction of NAD⁺ at 63 per cent the rate withL-proline. D-proline is not a substrate nor an inhibitor. L-prolineamide has 11 per cent the activity of L-proline and N-methyl-L-prolinehas a very slight activity. Other proline derivatives or thelower and higher homologues are completely inactive. Incubation with L-proline-¹⁴C in the presence of NAD⁺ yieldsone product which has a higher Rf than proline using butanol-aceticacid-water as the solvent in paper chromatography. Elution ofthis product and treatment with hydrogen peroxide gives severalproducts of high Rf with the same solvent mixture. None of theproducts is -aminobutyrate or glutamic acid. This eliminateseither P2C or P5C as the reaction product.