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Stability and Activation of Glutamate Apodecarboxylase from Pig Brain
Author(s) -
Porter Terence G.,
Martin David L.
Publication year - 1988
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1988.tb01173.x
Subject(s) - cofactor , enzyme , glutamate decarboxylase , chemistry , glutamate receptor , pyridoxal , dissociation constant , pyridoxal phosphate , kinetics , enzyme activator , biochemistry , dissociation (chemistry) , reaction rate constant , specific activity , biophysics , biology , receptor , physics , quantum mechanics
The stability and activation of glutamate apodecarboxylase was studied with three forms of the enzyme from pig brain (referred to as the α, β, and γ 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 β‐glutamate decarboxylase (β‐GAD) at 30°C, t l/2 values of apo‐ and holoenzyme were 17 and > 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 ( K r ) and apparent dissociation constant ( K D ) for the binding of pyridoxal‐P to the apoenzyme differed substantially among the forms (for α ‐, β ‐, and γ‐GAD, k r = 0.032, 0.17, and 0.27 min −1 , and K D = 0.014, 0.018, and 0.04 μ M ). ATP was a strong competitive inhibitor of activation ( K i = 0.45, 0.18, and 0.39 μ M for α‐, β‐, and γ‐GAD). In contrast, P i stimulated activation at 1–5 m M but inhibited at much higher concentrations. The results suggest that ATP is important in stabilizing the apoenzyme in brain and that ATP, P i , and other compounds regulate its activation.