Identity of proline dehydrogenase and delta1-pyrroline-5-carboxylic acid reductase in Clostridium sporogenes.
Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells.
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003013:Clostridium / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011392:Proline; D011762:Pyrroline Carboxylate Reductases / Q000302:isolation & purification / Q000378:metabolism*
D011392:Proline; D000594:Amino Acid Oxidoreductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011762:Pyrroline Carboxylate Reductases; D005947:Glucose