Correlating amino acid conservation with function in tyrosyl-tRNA synthetase

Sequence comparisons have been combined with mutational and kinetic analyse s to elucidate how the catalytic mechanism of Bacillus stearotheyl-nophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine subs trate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and tr ansfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Pr evious investigations indicate that the class I conserved KMSKS motif is in volved in only the first step of the reaction (i.e. tyrosine activation). H ere, 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 c onserved in a subset of the class I amino-acyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 a nd Arg86, stabilize the transition states for both steps of the tRNA aminoa cylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA syn thetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability o f 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 aci ds and other small molecules with ATP. (C) 2000 Academic Press.