Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: Identification of amino acid residues labeled by periodate-oxidized tRNA sup fMet molecules having modified lengths at the 3 prime -acceptor end

Journal Article · Tue Sep 04 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00487a029· OSTI ID:5707596

Hountondji, C; Schmitter, J M; Beauvallet, C; Blanquet, S ^[1]

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1. Laboratoire de Biochimie, Palaiseau (France)

Initiator tRNA molecules modified at the 3{prime}-end and lacking either A{sub 76} (tRNA-C{sub 75}), the C{sub 75}-A{sub 76} (tRNA-C{sub 74}), the C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 73}), or the A{sub 73}-C{sub 74}-C{sub 75}-A{sub 76} (tRNA-A{sub 72}) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTS{sub T}), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C{sub 75}ox, tRNA-A{sub 73}ox, and tRNA-A{sub 72}ox) abolished both the isotopic ({sup 32}P)PP{sub i}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-C{sub 75}ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C{sub 75}. The peptides of MTS{sub T} labeled with either tRNA-C{sub 75}ox or tRNA-C{sub 74}ox were identified. In a previous work all these peptides but one (peptide D) had been already found labeled upon MTS{sub T} incubation with ({sup 14}C)tRNA-A{sub 76}ox. According to the crystallographic structure of MTS{sub T}, the labeled residues K335, K61, K142, K147, and K149 are within a sphere of about 5.5-{angstrom} radius. The present results therefore argue for a marked flexibility of the 3{prime}-end of the enzyme-bound tRNA, enabling it to contact any of the identified reacting residues. Such a cluster of basic amino acids may reflect ionic requirements in the guiding of the negatively charged CCA arm of tRNA toward enzyme-bound methionyl-adenylate.

• OSTI ID: 5707596
• Journal Information: Biochemistry; (USA), Vol. 29:35; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LIGASES
MOLECULAR STRUCTURE
TRANSFER RNA
ELECTROPHORESIS
CARBON 14 COMPOUNDS
ESCHERICHIA COLI
METHIONINE
PEPTIDES
PHOSPHORUS 32
STOICHIOMETRY
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DRUGS
ENZYMES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RNA
550201* - Biochemistry- Tracer Techniques