Dimeric glutamyl-tRNA synthetases from wheat. Kinetic properties and functional structures

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Glutamyl-tRNA synthetases are crucial enzymes in protein synthesis.
• Understanding their kinetic properties and quaternary structure is essential for elucidating aminoacylation mechanisms.

Purpose of the Study:

• To determine the Michaelis constants (Km) for tRNA, glutamic acid, and MgATP for three dimeric glutamyl-tRNA synthetases (C, P, and E).
• To investigate the role of cognate tRNA in the [32P]PPi-ATP isotopic exchange reaction.
• To analyze the enzyme's active form and its dissociation equilibrium with monomers.

Main Methods:

• Enzyme kinetics assays were performed to measure Michaelis constants.
• Isotopic exchange reactions were used to assess enzyme activity in the presence and absence of tRNA.
• Enzyme activity was analyzed as a function of enzyme concentration to determine quaternary structure and dissociation constants.

Main Results:

• Michaelis constants for tRNA, glutamic acid, and MgATP were determined for synthetases C, P, and E.
• High MgATP concentrations (>2 mM) caused significant inhibition.
• Cognate tRNA was essential for [32P]PPi-ATP isotopic exchange, and hyperbolic saturation by glutamic acid was not observed without tRNA.
• Enzyme activity analysis indicated that active forms are dimers in equilibrium with inactive monomers.
• Dissociation constants (Kd) for the dimer-monomer equilibrium were found to be 43 nM, 53 nM, and 87 nM for synthetases C, P, and E, respectively.

Conclusions:

• Glutamyl-tRNA synthetases C, P, and E function as dimers in the tRNA aminoacylation reaction.
• The dimer-monomer equilibrium is a key feature of these enzymes' activity.
• Kinetic parameters provide insights into the substrate binding and catalytic mechanisms of these essential enzymes.