Time‐resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: Conformational analysis of intermediates and transition‐state complexes

Abstract

Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry30,3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the yglutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.Keywords: Escherichia coli; fluorescence studies; glutamine synthetase; transition-state complexes; tryptophan mutants Glutamine synthetase (GS) plays a central role in the nitrogen metabolism of both prokaryotes and eukaryotes, catalyzing the ATP-dependent condensation of glutamate with ammonia to form glutamine (Ginsburg, 1972;Stadtman & Ginsburg, 1974). The bacterial enzymes consist of a highly symmetrical dodecamer formed by two face-toReprint requests to: Joseph J. Villafranca, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.Abbreviations: W57L, the site-directed mutant that has Trp-57 replaced by leucine; W158S or WlSSF, the site-directed mutants that have Trp-158 replaced by serine or phenylalanine; glutamylphosphate-ADP-GS, the ternary complex formed after the addition of ATP and glutamate to glutamine synthetase.

Functional importance of Asp56 from the α‐polypeptide of Phaseolus vulgaris glutamine synthetase

“…Contrarily, the bacterial enzyme has been the subject of three‐dimensional structure determination for several bacterial sources, such as Salmonella typhimurium [5, 6] and Escherichia coli [7]. Mechanistic models for catalysis have been proposed [8–10], the models being consistent with results obtained by site‐directed mutagenesis studies [7, 11–13].…”

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy

“…The tryptophan mutant forms GS were constructed using site-directed mutagenesis techniques and kindly provided by Drs. William Atkins (Atkins & Villafranca, 1992) and Toru Maruyama of the Villafranca laboratory. Fully unadenylylated and adenylylated forms of wild-type and mutant GS were obtained by transforming the plasmid into E. coli strains YMC2 IE and YMC21D, respectively (Sambrook et al, 1989).…”