Substrate-induced conformational changes of the truncated catalytic domain of Geobacillus stearothermophilus lysyl-tRNA synthetase as examined by fluorescence.
The substrate-induced conformational change of the truncated C-terminal catalytic domain (CAT) of Geobacillus stearothermophilus lysyl-tRNA synthetase was examined by measuring tryptophan fluorescence of the truncated CAT domain in the presence or absence of the truncated N-terminal tRNA anticodon-binding domain (TAB). The fluorescence spectrum of CAT was not changed by the addition of l-lysine or ATP, whereas the intensity increased by adding a lysyl-adenylate analogue, suggesting that the CAT fluorescence increases when lysyl-adenylate is formed in the active site of CAT in l-lysine activation. In the presence of TAB, the addition of l-lysine to CAT decreased the fluorescence, and the subsequent addition of ATP recovered partially the decreased intensity, as is similar to the case of the intact enzyme. The static parameters of the CAT-TAB complex were similar to those of the intact enzyme, suggesting that a somewhat impaired structure of CAT is repaired on the formation of the complex with TAB. The mutational analysis of the fluorescence showed that Trp314 but not Trp332 is responsible for the observed fluorescence changes. The role of the TAB domain in the intact enzyme is considered to enhance the binding efficiency of lysyl-adenylate to the CAT domain.
Saruwatari
Yoshihiro
Y
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Wada
Takumi
T
Takita
Teisuke
T
Inouye
Kuniyo
K
D020134:Catalytic Domain; D005453:Fluorescence; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism* / Q000494:pharmacology; D008250:Lysine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding
D050505:Mutant Proteins; D008250:Lysine-tRNA Ligase; D008239:Lysine