Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: Exploration of the role of arginines 339 and 359 in activity

Abstract

In order to understand the nature of ATP and t-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg --t Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the K,,, values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine) (DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in K,,, value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kc,, than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar K,,, values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA). The R339C mutant, upon modification with BPA, had a greater kc,, than WT enzyme; however, the R359C mutant did not show significant regeneration of activity with this reagent. The data are consistent with X-ray crystallographic studies showing Arg 339 and Arg 359 at the active site of glutamine synthetase (Liaw SH, Eisenberg D, 1994, Biochemistry 33:675-681) interacting with ATP, glutamate, and intermediates along the catalytic pathway. Because enzyme activity could be restored for the R339C mutant by monofunctional (amine) and bifunctional (amidine) reagents, Arg 339 most likely interacts with substrates in a monodentate fashion. Conversely, Arg 359 seems to interact bifunctionally with substrates because covalent modification of R359C with BPA did not lead to a significant regain of catalytic activity.

Chemical Rescue of a Mutant Protein-tyrosine Kinase

“…As in other examples of chemical rescue, a high concentration is required for saturation of the active site with a small molecule, indicating that the affinity of the activator for the enzyme is relatively weak (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The K m values for ATP (K m ϭ 40 Ϯ 4 M) and poly(Glu,Tyr) (K m ϭ 115 Ϯ 10 g/ml) were only modestly affected by the addition of 50 mM guanidinium to R318A.…”

“…There are a number of reported examples where mutant, catalytically defective enzymes have been reactivated by introducing small molecules that have the requisite properties of the altered residues (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). By making slight structural changes in the small molecule activators, it is possible to learn more about the specific molecular contributions of the altered amino acid residue toward catalysis.…”

Identification of Three Key Active Site Residues in the C-terminal Domain of Human Recombinant Folylpoly-γ-glutamate Synthetase by Site-directed Mutagenesis

“…Hence, the effects of Asp-335 on binding of ATP and folate could be either direct or secondary to poor binding of K ϩ . Two arginine residues play key roles in bacterial GS (15); GS Arg-321 binds the ␣-carboxyl of the glutamic acid (the equivalent to the side chain of the folate substrate for FPGS), and structural and mutagenesis studies (15,18) identify S. typhimurium GS Arg-359 as a key residue for orientation of the ␥-carboxyl group of the glutamic acid and for stabilization of the mixed anhydride transition state intermediate. It would seem likely from our results that FPGS Arg-377 is involved in orientation of the incoming glutamic acid within the active site and might well interact with and stabilize the phosphorylated intermediate of the FPGS reaction, thus explaining the 95% drop in k cat in the R377A mutant.…”

Synthesis and characterization of supramolecular protein aggregates: self-assembled, molecularly-ordered, tubes from electrostatic complementation of glutamine synthetase dodecamers

“…Protein was expressed and purified as described previously , using the modifications described for mutants that do not effectively zinc precipitate. Chemical modification with CA or IA were accomplished as described previously (Dhalla et al, 1994;Greenhalgh et al, 1992). Briefly, 1 ml of a solution containing 1.0 mg protein/ml was incubated with 200 mM IA or CA in 50 mM HEPES, pH 7.2, 100 mM KCl, 1 mM MnCl 2 at room temperature for 3-4 h. Excess reagent was removed by gel filtration chromatography with Sephadex G-25.…”