Interaction of a new gamma-glutamyl-phosphate analog, 4-(phosphonoacetyl)-L-alpha-aminobutyrate, with glutamine synthetase enzymes from Escherichia coli, plant, and mammalian sources

The interactions of a newly synthesized nonreactive analog of γ-glutamyl-phosphate, 4-(phosphonoacetyl)-l-α-aminobutyrate, PALAB, have been studied with glutamine synthetases from bacterial, plant, and mammalian sources. The Escherichia coli enzyme fails to bind PALAB any more tightly than it does l-glutamate. In contrast, the pea seed enzyme is potently inhibited by PALAB, due to a two-step tight sequestering of PALAB into the active site and very slow release. The relative affinities of these two enzymes for transition state analogs with tetrahedral symmetry at position 5 (e.g., l-methionine-sulfoximine) were found previously to be the reverse of those for PALAB (F. C. Wedler and B. R. Horn, 1976, J. Biol. Chem.251, 7530–7538). Interestingly, the mammalian (ovine brain) enzyme exhibits no marked preference for PALAB vs l-methionine-sulfoximine, binding both reversibly about 10-fold tighter than l-glutamate. Noncompetitive kinetics for PALAB vs l-Glu with the ovine brain enzyme appear to arise from several effects: dissociation of PALAB that is much slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{s}$) than turnover, and preferential binding of PALAB to free E rather than E·ATP, whereas l-Glu prefers E·ATP over free E. The data strongly suggest that γ-Glu-phosphate, a likely intermediate in the reaction pathway, is stabilized in the active sites of these three enzymes to very different extents, implying that the chemical reaction profiles for each enzyme also differ considerably. Other known differences consistent with this hypothesis are: relative rates of PALAB association and dissociation, substrate binding order, the occurrence of isotopic exchanges with partial reaction systems or of analog-induced conformational changes, and different mechanistic roles for divalent metal ions in catalysis.