Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase.
Residue Thr-51 at the active site of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) has been replaced with all the smaller amino acids by protein engineering to investigate direct and indirect effects of mutation on substrate binding and catalysis. The gamma-hydroxyl group of Thr-51 was thought to be 0.5 A too far from the ribose ring oxygen of ATP to form a hydrogen bond. Consistent with this, it is found that mutation of Thr-51----Cys-51, which should place the gamma-thiol group within its correct distance for hydrogen bonding, increases the affinity of the enzyme for ATP. Other mutations (Ser-51, Ala-51, and Gly-51) show the contributions to binding of the other atoms in the side chain of Thr-51. A family of enzymes has been produced, TyrTS(Thr-51) (wild type), TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51), in which the value of kcat/KM for ATP in aminoacylation increases along the series. This is achieved by the value of KM decreasing significantly (2.5, 1.25, 0.29, and 0.019 mM, respectively) while there are smaller decreases in kcat (4.7, 4.0, 2.9, and 1.8 s-1, respectively). These variations cause each one of the enzymes to be more active than the others at particular concentrations of ATP. For example, at concentrations of ATP greater than 5.9 mM, TyrTS(Thr-51) is the most active, while TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51) are the most active at 5.9-2.2, 2.2-0.42, and less than 0.42 mM ATP, respectively. Interestingly, position 51 shows variation in tyrosyl-tRNA synthetases isolated from different organisms.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001665:Binding Sites; D011756:Diphosphates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D006860:Hydrogen Bonding; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D013816:Thermodynamics; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism
D011756:Diphosphates; D012346:RNA, Transfer, Amino Acyl; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase