Inhibition of Glucuronokinase by Substrate Analogs

Abstract

Glucuronokinase from Llum lougMorum polen was purified 30-to 40-fold on a blue dextran-Sepharose column. Substrate analogs were tested for inhibitory effects, and nucleotide substrate specificity of the enzyme was determined. Nine nucleotides were tested, and al were inhibitory when the substrate was ATP. ADP was competitive with ATP and had a Ki value of 0.23 mm. None of the other nucleotide triphosphates could effectively substitute for ATP as a nucleotide substrate. Ten mm dATP and ITP reacted only 3% as rapidly as 10 mM ATP, while the rates for 10 mM GTP, CTP, UTP, and TTP were less than 1%. The glucuronic acid analogs, methyl a-glucuronoside, methyl 8-glucuronoside, 8-glucuronic acid-i-phosphate, and 4-0-methylglucuronic acid were tested as possible enzyme inhibitors. The three methyl derivatives showed little or no inhibition. The ,8-glucuronic acid-i-phosphate was inhibitory, with 50% inhibition obtained at 1 to 3 mm depend on the concentration of the glucuronic acid. It is concluded that the glucuronic acid-binding site on the enzyme is highly selective.Glucuronokinase (EC 2.7.1.43) catalyzes the reaction glcUA2 + ATP --a-glcUA-1-P + ADP (13) and is an enzyme of the myoinositol oxidation pathway (4, 8). The enzyme is inhibited by the pathway intermediates, a-glcUA-I-P and UDP-glcUA, suggesting it may have a regulatory role in the formation of cell wall uronic acids and pentoses (7). A number of sugars and sugar acids were evaluated as enzyme inhibitors, but none were effective (5, 7). Derivatives of glcUA have not been tested and there is no information concerning the possible inhibitory effects of nucleotides.The present work deals with the selectivity of glucuronokinase toward glucuronic acid analogs and nucleotides other than ATP. The enzyme was purified by a new one-step procedure which utilized affinity chromatography on blue dextran-Sepharose resin. MATERIALS AND METHODSReagents. The nucleotides, the CNBr-activated Sepharose, Escherichia coli /3-glucuronidase (type VII), calf intestine alkaline phosphatase (type I), and fl-glucose-1-P dicyclohexylammonium salt were obtained from Sigma Chemical Co for 10 min. The supernatant was decanted through two layers of cheesecloth and used as the crude enzyme preparation. The crude enzyme was purified on a blue dextran-Sepharose affinity column. The column (55 x 6 mm) was loaded with 45 to 55 ml of crude enzyme preparation (-8 mg of protein/ml) at a peristaltic pump flow rate of 0.4 ml/min. The column was washed with 50 ml of 10 mm HEPES (pH 7.2) containing 1 mm DTT. The enzyme was eluted from the column with the same buffer containing 100 mm KCI. The KCI eluate was made up to 50 ml with wash buffer and concentrated to less than I ml using an Amicon XM-50 ultrafilter. The salts were further removed by reconstituting the concentrate to 50 ml with the wash buffer and again concentrating it to I ml. The second concentrate was divided into 100-,ul portions and stored in liquid N2. Under these storage conditions no loss of enzyme activity was observed over...

Generation of nucleotide sugars for biomass formation in plants

“…Phosphorylation of GlcA also constitutes the myoinositol oxidation (MIO) pathway through which UDPGlcA is generated from myo-inositol via GlcA and GlcA 1-P. Glucuronokinase activity was first found in a soluble fraction of mung bean seedlings in 1959 (Neufeld et al 1959), and also reported in lily (Lilium longiflorum) pollens (Gillard and Dickinson 1978;Leibowitz et al 1977). Recently, Pieslinger et al purified the enzyme from lily pollens, and identified GlcAK gene encoding glucuronokinase in Arabidopsis (Pieslinger et al 2009(Pieslinger et al , 2010.…”