Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.
Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.
Takita
Teisuke
T
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan. takita@kais.kyoto-u.ac.jp
Nakagoshi
Makoto
M
Inouye
Kuniyo
K
Tonomura
Ben'ichiro
B
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
D011994:Recombinant Proteins; D014364:Tryptophan; D008250:Lysine-tRNA Ligase; D008239:Lysine