Interactions between Escherichia coli arginyl-tRNA synthetase and its substrates

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Arginyl-tRNA synthetase (ArgRS) is crucial for protein synthesis, catalyzing the attachment of arginine to its cognate tRNA.
• Understanding enzyme-substrate interactions is key to elucidating catalytic mechanisms and regulation.

Purpose of the Study:

• To investigate the intricate binding interactions between Escherichia coli arginyl-tRNA synthetase and its substrates: arginine, ATP, and tRNA.
• To determine how the presence of one substrate affects the binding affinity of others to the enzyme.

Main Methods:

• Equilibrium dialysis
• Fluorescence titration
• Enzyme protection against heat inactivation

Main Results:

• Individual substrate dissociation constants were determined: arginine (~70 µM), ATP (~0.85 mM), and tRNA (~0.45 µM).
• Arginine binding affinity significantly increased (KD ~16 µM) in the presence of saturating ATP and tRNA, approaching the Km for aminoacylation (~12 µM).
• ATP binding affinity decreased in the presence of tRNA (KD ~3 mM), while tRNA binding remained unaffected by single substrates.

Conclusions:

• Escherichia coli arginyl-tRNA synthetase displays pronounced substrate cooperation, where the simultaneous presence of ATP and tRNA enhances arginine binding.
• These findings highlight enzyme-mediated interactions between substrates under catalytic conditions, influencing enzyme efficiency.
• The enzyme's ability to bind tRNA was not compromised by periodate oxidation, suggesting structural integrity for substrate recognition.