Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase
Ambasht, P.K.; Malhotra, O.P.; Kayastha, A.M.
Indian Journal of Biochemistry and Biophysics 34(4): 365-372
1997
ISSN/ISBN: 0301-1208 PMID: 9491646 Document Number: 475921
Pyruvate kinase was purified from mung bean [Vigna radiata], and the effects on enzyme activity of various metabolites of the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as nucleotides and amino acids, were studied. Active site groups were also investigated. AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, 3-phosphoglycerate, isocitrate, malate and alpha -ketoglutarate had no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. ATP, GTP and UMP were weakly inhibitory. Moderate to strong inhibition was observed with citrate, succinate, glutamate and oxalate. Inhibition brought about by ATP and citrate when present together showed a synergistic effect. Inhibition by citrate was non-competitive with respect to both phosphoenolpyruvate (PEP) and ADP, suggesting the presence of a regulatory site. Low concentrations of iodoacetamide (0.1 and 0.2 mM), N-ethylmaleimide (0.05-0.1 mM) and p-chloromercuribenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in the presence of methylene blue (100 and 200 micro g/ml) and rose bengal (5 and 10 micro g/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate (DEP), a time dependent exponential decay in its activity was observed with a parallel increase in absorbance at 240 nm. PEP protected the enzyme against inactivation by DEP. It is concluded that SH and imidazole groups are vital for the activity of the enzyme.