Investigation of transition-state stabilization by residues histidine-45 and threonine-40 in the tyrosyl-tRNA synthetase.
We have analyzed various mutations involving residues Thr-40 and His-45 in the tyrosyl-tRNA synthetase of Bacillus stearothermophilus. The utilization of binding energy in catalysis of tyrosyl adenylate formation from tyrosine and ATP was determined from the free energy profiles for the mutant enzymes. Our results confirm that the side chains of Thr-40 and His-45 provide a binding site for the pyrophosphoryl portion of the transition state of this reaction and for pyrophosphate in the reverse reaction. Deletion of these side chains destabilizes the transition-state by 4.9 and 4.1 kcal mol-1, respectively, consistent with a charged hydrogen-bonding interaction. To examine the role of His-45 further, we constructed the potentially conservative mutations His----Gln-45 and His----Asn-45. Both mutant enzymes are debilitated compared with the native enzyme. The His----Gln-45 enzyme is more active than enzyme in which the complete side chain is deleted (His----Ala-45), and so in this location glutamine is a semiconservative replacement. In contrast, the His----Asn-45 mutation is significantly worse than simple deletion of the side chain, indicating that asparagine at this position causes active destabilization of the transition state compared to His----Ala-45. The amide-NH2 of glutamine may be considered stereochemically equivalent to the epsilon-NH of histidine whereas the amide-NH2 of asparagine is comparable to the delta-NH of histidine. The results suggest that the epsilon-NH rather than the delta-NH group of His-45 is involved in the transition-state stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013912:Threonine; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*
D013912:Threonine; D006639:Histidine; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase