Abstract
In order to understand the nature of ATP and L‐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 → Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the K~m~ 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~m~ value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater k~cat~ 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~m~ 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 k~cat~ 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.