REGENERATION OF CATALYTIC ACTIVITY OF GLUTAMINE-SYNTHETASE MUTANTS BYCHEMICAL ACTIVATION - EXPLORATION OF THE ROLE OF ARGININE-339 AND ARGININE-359 IN ACTIVITY

In order to understand the nature of ATP and L-glutamate binding to gl utamine synthetase, and the involvement of Arg 339 and Arg 359 in cata lysis, these amino acids were changed to cysteine via site-directed mu tagenesis. Individual mutations (Arg --> Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the K(m) val ues for the substrates ATP and glutamate were elevated substantially a bove the values for wild-type (WT) enzyme. Each cysteine was in turn c hemically 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) (produ cing a disulfide) were the reagents used to effect these chemical tran sformations. Upon reaction with CA, both R339C and R359C mutants showe d a significant regain of catalytic activity (50% and 70% of WT, respe ctively) 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 glu tamine 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 a nd glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothrei tol, as expected for a modified enzyme containing a disulfide bond. Mo dification of each cysteine-containing mutant to a lysine ''analog'' w as 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 wit h this reagent. The data are consistent with X-ray crystallographic st udies showing Arg 339 and Arg 359 at the active site of glutamine synt hetase (Liaw SH, Eisenberg D, 1994, Biochemistry 33:675-68 1) interact ing with ATP, glutamate, and intermediates along the catalytic pathway . Because enzyme activity could be restored for the R339C mutant by mo nofunctional (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 cov alent modification of R359C with BPA did not lead to a significant reg ain of catalytic activity.