TIME-RESOLVED FLUORESCENCE AND COMPUTATIONAL STUDIES OF ADENYLYLATED GLUTAMINE-SYNTHETASE - ANALYSIS OF INTERSUBUNIT INTERACTIONS

Adenylylation of Tyr-397 of each subunit of Escherichia coli glutamine synthetase (GS) down-regulates enzymatic activity in vivo. The overal l structure of the enzyme consists of 12 subunits arranged as two hexa mers, face to face. Research reported in this paper addresses the ques tion of whether the covalently attached adenylyl group interacts with neighboring amino acid residues to produce the regulatory phenomenon. Wild-type GS has two Trp residues (positions 57 and 158) and the adeny lylation site lies within 7-8 angstrom of the Trp-57 loop in the adjac ent subunit of the same hexameric ring; Trp-158 is about 35 k from the site of adenylylation. Fluorescence lifetimes and quantum yields have been determined for two fluorophores with wild-type and mutant GS. On e fluorophore is epsilon-AMP adenylylated GS (at Tyr-397), and the oth er fluorophore is the intrinsic protein residue Trp-57. These experime nts were conducted in order to detect possible intersubunit interactio ns between adenylyl groups and the neighboring Trp-57 to search for a role for the Trp-57 loop in the regulation of GS. The fluorescence due to epsilon-AMP of two adenylylated enzymes, wild-type GS and the W158 F mutant, exhibits heterogeneous decay kinetics; the data adequately f it to a double exponential decay model with recovered average lifetime values of 18.2 and 2.1 ns, respectively. The pre-exponential factors range from 0.66 to 0.73 for the long lifetime component, at five emiss ion wavelengths. The W57L-epsilon-AMP enzyme yields longer average lif etime values of 19.5 and 2.4 ns, and the pre-exponential factors range from 0.82 to 0.85 for the long lifetime component. An additional resi due in the Trp-57 loop, Lys-58, has been altered and the K58C mutant e nzyme has been adenylylated with epsilon-AMP on Tyr-397. Lys-58 is nea r the ATP binding site and may represent a link by which the adenylyl group controls the activity of GS. The fluorescence of epsilon-AMP-ade nylylated K58C mutant GS is best described by a triple exponential dec ay with average recovered lifetime values of 19.9, 4.6, and 0.58 ns, w ith the largest fraction being the median lifetime component. Relative quantum yields of epsilon-AMP-Tyr-397 were measured in order to deter mine if static quenching occurs from adenine-indole stacking in the wi ld-type GS. The relative quantum yield of the epsilon-AMP-adenylylated W57L mutant is larger than the wild-type protein by the amount predic ted from the difference in lifetime values: thus, no static quenching is evident. Intrinsic tryptophan fluorescence was also studied in the presence and absence of covalently attached adenylyl groups. The fluor escence decay parameters of Trp-57 are not significantly affected by t he presence of epsilon-AMP or AMP attached to Tyr-397. Enzymatic activ ity of the mutant proteins was also studied. The Mg-activated enzymes are nearly completely inhibited upon adenylylation, and regulation is not affected by mutation at Trp-57 or Lys-58. These results indicate t hat the Trp-57 loop dynamically interacts with the adenylyl group, but a ''ring stacked'' complex is not formed and is not a structural feat ure of the regulatory mechanism. This conclusion was further corrobora ted by computational minimization and molecular dynamics studies of GS with an AMP moiety built onto Tyr-397. Relative energies were sampled at various points as the Tr-57 ring and urine ring of AMP were target ed toward one another, the endpoint being a 3-angstrom parallel and st acked conformation. Dynamics simulations were performed with the paral lel, stacked conformation, as well as an extended conformation, as the starting point. All studies indicate that the stacked indole-purine c onformation is not favorable; however, a potential hydrogen bonding in teraction between the amine nitrogen of the tryptophan and the ring ox ygen of ribose is implied by dynamics simulations for certain conforma tions of the loop.