Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: identification of amino acid residues labeled by periodate-oxidized tRNA(fMet) molecules having modified lengths at the 3'-acceptor end.
Initiator tRNA molecules modified at the 3'-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopic [32P]PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75. The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified. The chymotryptic digestion of the covalent MTST.[14C]tRNA-C75ox complex yielded four peptides (A-D). In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified. Peptides A, B, C, and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Val141-Tyr165, and Glu433-Phe437, respectively, in the MTST primary structure.(ABSTRACT TRUNCATED AT 250 WORDS)
Hountondji
C
C
Laboratoire de Biochimie (URA CNRS 240), Ecole Polytechnique, Palaiseau, France.
Schmitter
J M
JM
Beauvallet
C
C
Blanquet
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis*; D012358:RNA, Transfer, Met / Q000378:metabolism*
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D008718:Methionine-tRNA Ligase