Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

Area of Science:

• Plant biochemistry
• Enzymology
• Sulphur metabolism

Background:

• ATP sulphurylase is a key enzyme in the assimilation of inorganic sulphur.
• Understanding its activity and substrate specificity is crucial for plant physiology.
• Previous studies have indicated its role in sulphate activation.

Purpose of the Study:

• To purify and characterize ATP sulphurylase from spinach leaf tissue.
• To investigate the enzyme's kinetic properties and substrate preferences.
• To explore the enzyme's role in the synthesis of adenosine 5'-sulphatophosphate.

Main Methods:

• Purification of ATP sulphurylase from spinach leaf extracts.
• Enzyme activity assay using sulphate-dependent [32P]PP(i)-ATP exchange.
• Kinetic analysis (Km, Vmax) with various substrates including ATP, dATP, sulphate, and selenate.
• Demonstration of adenosine 5'-[(35)S]sulphatophosphate synthesis using coupled enzyme reactions.

Main Results:

• ATP sulphurylase was purified 1000-fold, showing no ADP sulphurylase activity.
• The enzyme exhibited optimal activity at pH 7.5-9.0, requiring Mg(2+) or Co(2+).
• Sulphate and ATP were substrates with Km values of 3.1 mM and 0.35 mM, respectively.
• dATP and selenate were also utilized as substrates, albeit with lower efficiency.
• Adenosine 5'-sulphatophosphate synthesis was confirmed in a coupled reaction system.

Conclusions:

• Spinach ATP sulphurylase is a distinct enzyme with specific kinetic properties.
• The enzyme demonstrates flexibility in utilizing dATP and selenate, impacting sulphur assimilation pathways.
• This study provides detailed characterization of ATP sulphurylase, essential for understanding plant sulphur metabolism.