ATP binding to brain l-glutamate decarboxylase: A study by affinity chromatography

Area of Science:

• Biochemistry
• Enzymology
• Neuroscience

Background:

• l-glutamate decarboxylase (GAD) is a key enzyme in GABA synthesis.
• Understanding cofactor and substrate binding is crucial for enzyme function.
• ATP is an essential molecule in cellular processes, but its direct interaction with GAD was not fully elucidated.

Purpose of the Study:

• To investigate the specific binding interaction between ATP and mouse brain GAD.
• To determine the binding site and affinity of ATP on GAD.
• To explore the relationship between ATP binding and pyridoxal 5?-phosphate (PLP) binding.

Main Methods:

• ATP-agarose affinity chromatography was employed using partially purified mouse brain GAD.
• Enzyme binding and dissociation were tested using varying concentrations of free ATP, ADP, and PLP.
• Kinetic analysis was performed to assess the effect of ATP binding on PLP interaction.

Main Results:

• GAD exhibited high-affinity binding to ATP immobilized via the N(6)-amino group on agarose.
• ATP binding was specific, with no significant binding observed when ATP was attached via ribosyl hydroxyls.
• Bound GAD could be eluted by free ATP but not by ADP, and PLP did not compete for the ATP binding site.
• ATP binding increased the enzyme's K(m) for PLP, indicating an allosteric effect.

Conclusions:

• Mouse brain GAD possesses a specific binding site for ATP, separate from the PLP binding site.
• The N(6)-amino group of ATP is critical for its high-affinity interaction with GAD.
• ATP binding influences the enzyme's interaction with its cofactor PLP, suggesting a regulatory mechanism.