Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis

Abstract

y-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified-y-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of-y-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 x 10-M. Enzyme activity was insensitive to the glutamate analog Lmethionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a y-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline. Proline biosynthesis in Escherichia coli occurs through a series of three enzymatic reactions catalyzed by the gene products of the proB, proA, and proC loci: through feedback inhibition of-y-glutamyl kinase by proline (1, 3). E. coli mutants which excrete proline can be obtained by selection for'resistance to the toxic proline analog 3,4-mg2 L-glutamate + ATP-)-y-glutamyl phosphate + ADP phosphate+ NADPH proA ,y-glutamyl phosphate + NADPH L-glutamic acid 5-semialdehyde + NADP+ + Pi L-glutamic acid 5-semialdehyde) A1-pyrroline-5-carboxylate roC P+ A1pyrroline-5-c'arboxylate + NADPH-L-proline + NADP' Reaction 1 is catalyzed by-y-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11) and most likely results in the production of an extremely labile intermediate, y-glutamyl phosphate (1, 8). Due to the lability of this compound, it has been proposed that-y-glutamyl kinase functions in a complex with the second enzyme of the pathway,-y-glutamyl phosphate reductase (1) (GP-reductase; L-glutamate 5-semialdehyde:NADP+ oxidoreductase [phosphorylating], EC 1.2.1.41). The product of reaction 2, Lglutamic acid 5-semialdehyde, undergoes a spontaneous cyclization to A-pyrroline-5-carboxylate, which is converted to proline through the action A1-pyrroline-5-carboxylate reductase (L-proline:NAD[P]+ 5-oxidoreductase, EC 1.5.1.2), the third enzyme in the pathway (1, 22). The second and third enzymes of proline biosynthesis have been purified and characterized (5, 9, 19); however, the first enzyme,-y-glutamyl kinase, has only been studied in cell-free extracts or crude preparations (1, 2). Regulation of proline biosynthesis is thought to be exerted primarily

Enhanced and feedback-resistant γ-glutamyl kinase activity of an Escherichia coli transformant carrying a mutated proB gene of Streptococcus thermophilus

“…A slight increase of Q-glutamyl-phosphate reductase speci¢c activity (from 5.1 to 8.0 nkat mg 31 ) was also observed, although no mutation in the proA gene was present. This was probably due to the occurrence of a physical interaction between the two enzymes, observed in other organisms [21], but the possibility of a positive downstream e¡ect of the proB mutation on proA transcription cannot be excluded.…”

Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation

“…The kinetic properties of the E. coli GK have been extensively characterized. The enzyme shows positive cooperativity with increasing glutamate concentration (Smith, Deutch & Rushlow, ; Seddon, Zhao & Meister, ): the concentration of glutamate that gives 50% activity ( S 0.5 ) in the absence of proline has been reported to be in the range of 16 mM (Seddon et al , ) to 82 mM (Pérez‐Arellano, Rubio & Cervera, ; Pérez‐Arellano et al , ). The latter researchers found that the S 0.5 for glutamate increased linearly with proline concentration, suggesting that glutamate and proline compete with each other for binding to the enzyme (Pérez‐Arellano et al , ).…”

Analysis of theEscherichia coli proBAlocus by DNA and protein sequencing

“…We selected for a spontaneous mutation, mapping within the proB locus of pLC7-19, which conferred cellular resistance to 100 ig/ml of DHP. GK activity, from strains harboring plasmids which conferred DHP resistance, was considerably more resistant to the inhibitory effects of proline than the GK activity from wild-type strains (9). A 3.0 kb Pstl DNA fragment, isolated from the modified pLC7-19 plasmid, was identified as encoding both the affected proB locus and the wild-type proA locus (10).…”