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Biosynthesis of proline in Pseudomonas aeruginosa. Properties of ;-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase

  • Krishna, Rangachar V. Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland
  • Beilstein, Paul Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland
  • Leisinger, Thomas Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland
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  • Biochemical Journal. - Portland Press Ltd.. - 1979, vol. 181, no. 1, p. 223-230
English γ-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from γ-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)+. The apparent Km values were: NAD+, 0.36mm; NADP+, 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP+ and 8mm with NAD+; Pi, 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of γ-glutamyl phosphate, inhibited this enzyme competitively (Ki=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH4)2SO4 fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (Km=0.05mm) was a better substrate than NADPH (Km=0.02mm). The apparent Km values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD+ at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)+, heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.
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