PROPERTIES OF BRAIN l‐GLUTAMATE DECARBOXYLASE: INHIBITION STUDIES1
Authors: Wu J.‐Y. , Roberts E. 
Published: October 2006

—l‐Glutamate decarboxylase purified from mouse brain was found to be highly sensitive to the sulfhydryl reagents, 5,5‐dithiobis (2‐nitrobenzoic acid) (DTNB) and p‐chloromerburibenzoate (PCMB), which were competitive inhibitors (Ki for DTNB is 1·1 · 10−8m). Iodoacetamide and iodoacetic acid were less effective inhibitors than DTNB and PCMB. The mercapto acids, 3‐mercaptopropionic, 2‐mercaptopropionic, and 2‐mercaptoacetic acids were potent competitive inhibitors with Ki values of 1·8, 53 and 300 μm, respectively. 2‐Mercaptoethanol was less effective. Aminooxyacetic acid was the most potent carbonyl‐trapping reagent tested inhibiting the enzyme activity completely at 1·6 μm, followed by hydroxylamine, hydrazine, semicarbazide, and d‐penicillamine. Carboxylic acids with a net negative charge were strong competitive inhibitors e.g. d‐glutamate (Ki 0·9 mm), α‐ketoglutarate (Ki, l·2mm), fumarate (Ki,1·8 mm), dl‐β‐hydroxyglutamate (Ki, 2·8 mm), l‐aspartate (ki, 3·1 mm) and glutarate (Ki, 3·5 mm). 2‐Aminophosphonobutyric and 2‐aminophosphonopropionic acids, phosphonic analogs of glutamate and aspartate, respectively, had no effect at l0mm. γ‐Aminobutyric acid, l‐glutamine, l‐γ‐methylene‐glutamine, and α,γ‐diaminoglutaric acid, amino acids with no net negative charge at neutral pH, had no effect at 5 mm. Glutaric and α‐ketoglutaric acids were the most potent inhibitors among the various dicarboxylic and α‐keto‐dicarboxylic acids tested (Ki, 3·5 and 1·2 mm, respectively). Compounds with one carbon less, succinic and oxalacetic acids, or with one carbon more, adipic and α‐ketoadipic acids, were less inhibitory. The monovalent cations, Li+, Na+, NH4+, and Cs+ had no effect on l‐glutamate decarboxylase activity in concentrations up to 10mm. Divalent cations, on the other hand, were very potent inhibitors. Among eleven divalent cations tested, Zn2+ was the most potent inhibitor, inhibiting to the extent of 50 per cent at 10μm. The decreasing order of inhibitory potency was: Zn2+ > Cd2+, Hg2+, Cu2+ > Ni2+ > Mn2+ Co2+ > Ba2+ > Ca2+ > Mg2+ > Sr+2, The anions, I−, Br−, Cl− and F− were only weak inhibitors. The Ki value for Cl− was 17mm. The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme. Intermediates of glycolysis had little effect on l‐glutamate decarboxylase activity, but intermediates of the tricarboxylic acid cycle, e.g. α‐ketoglutarate (Ki= 1·2 mm) and fumarate (Ki= 1·8 mm) were relatively potent inhibitors. The nucleotides, ATP, ADP, AMP, cyclic AMP, GTP, GDP, GMP, and cyclic GMP were weak inhibitors. l‐Norepinephrine (Ki= 1·3 mm) and serotonin were potent inhibitors, while acetylcholine, dopamine and histamine were less effective. Ethanol and dioxane inhibited the enzyme activity to the extent of 20‐50 per cent at 10 per cent (v/v), while slight activation was observed at low concentrations (0·1‐1 per cent) of both solvents. The possible role of Zn2+ and some metabolites in the regulation of steady‐state levels of γ‐aminobutyric acid also was discussed.