Purification and Properties of the Bifunctional Proline Dehydrogenase/1‐Pyrroline‐5‐Carboxylate Dehydrogenase from Pseudomonas aeruginosa
- 1 December 1982
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 129 (1), 67-75
- https://doi.org/10.1111/j.1432-1033.1982.tb07021.x
Abstract
Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the 2 consecutive reactions of the oxidation of proline to glutamic acid, was purified from P. aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-.DELTA.1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (> 98% pure). The MW of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a MW of 242,000; polyacrylamide gel electrophoresis under native conditions led to the appearance of 2 catalytically active forms of the enzyme with MW of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and .DELTA.1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for .DELTA.1-pyrroline-5-carboxylic acid was nonhyperbolic.This publication has 34 references indexed in Scilit:
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