title: Comparison of the catalytic roles played by the KMSKS motif in the human and Bacillus stearothermophilus trosyl-tRNA synthetases.
abstract: The Class I aminoacyl-tRNA synthetases are characterized by two signature sequence motifs, "HIGH" and "KMSKS." In Bacillus stearothermophilus tyrosyl-tRNA synthetase, the KMSKS motif (230KFGKT234) has been shown to stabilize the transition state for tyrosine activation through interactions with the pyrophosphate moiety of ATP. In most eukaryotic tyrosyl-tRNA synthetases, the second lysine in the KMSKS motif is replaced by a serine or an alanine residue. Recent kinetic studies indicate that potassium functionally compensates for the absence of the second lysine in the human tyrosyl-tRNA synthetase (222KKSSS226). In this paper, site-directed mutagenesis and pre-steady state kinetics are used to determine the roles that serines 224, 225, and 226 play in catalysis of the tyrosine activation reaction. In addition, the catalytic role played by a downstream lysine conserved in eukaryotic tyrosyl-tRNA synthetases, Lys-231, is investigated. Replacing Ser-224 and Ser-226 with alanine decreases the forward rate constant 7.5- and 60-fold, respectively. In contrast, replacing either Ser-225 or Lys-231 with alanine has no effect on the catalytic activity of the enzyme. These results are consistent with the hypothesis that the KMSSS sequence in human tyrosyl-tRNA synthetase stabilizes the transition state for the tyrosine activation reaction by interacting with the pyrophosphate moiety of ATP. In addition, although they play similar roles in catalysis, the overall contribution of the KMSKS motif to catalysis appears to be significantly less in human tyrosyl-tRNA synthetase than it is in the B. stearothermophilus enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Austin
authors.forename: Eric A
authors.initials: EA
authors.lastname: First
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D012343:RNA, Transfer / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D012694:Serine; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D014448:Tyrosine-tRNA Ligase; D008239:Lysine; D011188:Potassium