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

The binding of ATP to brain l-glutamate decarboxylase (GAD) was studied by means of ATP-agarose chromatography, utilizing partially purified GAD from mouse brain after DEAE-cellulose chromatography and ammonium sulfate fractional precipitation. GAD was found to bind with a high affinity to the ATP-agarose with the ATP molecule linked to the beaded agarose through the N⁶-amino group. Agarose with ATP attached through the ribosyl hydroxyls was totally ineffective to bind the enzyme. GAD bound to the immobilized ATP could be dissociated by free ATP (10–50 mM), but not by ADP at a concentration as high as 100 mM. Mg²⁺ was not a required factor for the binding. The enzyme binding to the ATP-agarose occurred under a saturating concentration (50 μM) of pyridoxal 5′-phosphate (PLP). Moreover, GAD bound to the ATP-agarose was not dissociated by PLP even at 1.0 mM, indicating no competition of PLP with ATP for the same binding site on the enzyme. Kinetic characterization showed that binding of ATP raised the K_m of the enzyme for PLP. Our approach provides direct evidence that there is a specific binding site on GAD for ATP, which is distinct from the binding site for PLP.