Correlating amino acid conservation with function in tyrosyl-tRNA synthetase.
Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.
Xin
Y
Y
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, 71130, USA.
Li
W
W
Dwyer
D S
DS
First
E A
EA
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics; D004795:Enzyme Stability; D019143:Evolution, Molecular; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012346:RNA, Transfer, Amino Acyl; D013912:Threonine; D014443:Tyrosine; D006639:Histidine; D000255:Adenosine Triphosphate; D001120:Arginine; D014448:Tyrosine-tRNA Ligase; D008239:Lysine