Stability and activation of glutamate apodecarboxylase from pig brain.
The stability and activation of glutamate apodecarboxylase was studied with three forms of the enzyme from pig brain (referred to as the alpha, beta, and gamma forms). Apoenzyme was prepared by incubating the holoenzyme with aspartate followed by chromatography on Sephadex G-25. Apoenzyme was much less stable than holoenzyme to inactivation by heat (for beta-glutamate decarboxylase (beta-GAD) at 30 degrees C, t1/2 values of apo- and holoenzyme were 17 and greater than 100 min). ATP protected holoenzyme and apoenzyme against heat inactivation. The kinetics of reactivation of apoenzyme by pyridoxal-P was consistent with a two-step mechanism comprised of a rapid, reversible association of the cofactor with apoenzyme followed by a slow conversion of the complex to active holoenzyme. The reactivation rate constant (kr) and apparent dissociation constant (KD) for the binding of pyridoxal-P to apoenzyme differed substantially among the forms (for alpha-, beta-, and gamma-GAD, kr = 0.032, 0.17, and 0.27 min-1, and KD = 0.014, 0.018, and 0.04 microM). ATP was a strong competitive inhibitor of activation (Ki = 0.45, 0.18, and 0.39 microM for alpha-, beta-, and gamma-GAD). In contrast, Pi stimulated activation at 1-5 mM but inhibited at much higher concentrations. The results suggest that ATP is important in stabilizing the apoenzyme in brain and that ATP, Pi, and other compounds regulate its activation.
Porter
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Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Martin
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001224:Aspartic Acid / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D005968:Glutamate Decarboxylase / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013552:Swine
D001051:Apoenzymes; D001059:Apoproteins; D004793:Enzyme Reactivators; D010710:Phosphates; D001224:Aspartic Acid; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D005968:Glutamate Decarboxylase