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

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Abstract
.gamma.-Glutamyl kinase, the 1st enzyme of the proline biosynthetic pathway, was purified to homogeneity from an E. coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native MW of 236,000 and was comprised of 6 identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified .gamma.-glutamyl phosphate reductase, the 2nd enzyme of the proline pathway. Plots of .gamma.-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 .times. 10-4 M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a .gamma.-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. 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.