Tyrosyl-tRNA synthetase from wheat germ

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Tyrosyl-tRNA synthetase (TyrRS) is crucial for protein synthesis, catalyzing the aminoacylation of tRNA with tyrosine.
• Understanding plant enzyme characteristics is vital for molecular biology and agricultural applications.

Purpose of the Study:

• To purify and characterize tyrosyl-tRNA synthetase (TyrRS) from wheat germ extract.
• To investigate the kinetic properties and quaternary structure of wheat germ TyrRS.
• To identify and differentiate potential isoenzymes of TyrRS in wheat germ.

Main Methods:

• Enzyme purification using ultracentrifugation, ammonium acetate precipitation, and column chromatography.
• SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to determine subunit molecular weight.
• Gel filtration chromatography to assess native molecular weight and quaternary structure.
• Enzyme kinetics assays to determine optimal conditions (Mg2+, ATP, pH, KCl) and kinetic parameters (Km).

Main Results:

• TyrRS was purified 5,000-fold, exhibiting a native molecular weight of 110,000 Da, suggesting an alpha 2 dimeric structure.
• Optimal activity was observed at 6 mM Mg2+, 4 mM ATP, 200 mM KCl, and pH 8.
• Km values for ATP, tyrosine, and tRNA were determined to be 40, 3.3, and 1.5 microM, respectively.
• A second tyrosylating activity was detected, specific for Escherichia coli tRNA, suggesting it is wheat germ mitochondrial TyrRS.

Conclusions:

• The primary wheat germ TyrRS is a dimeric enzyme with specific kinetic requirements.
• The identification of a distinct mitochondrial TyrRS highlights compartmentalization of aminoacyl-tRNA synthetases in plants.
• Further studies are needed to explore the instability of TyrRS activity and stabilization methods.