Regulatory properties and active site groups of cytosolic mung bean pyruvate kinase.
Properties of mung bean pyruvate kinase were studied and the active site groups were derived. Metabolites like AMP, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose-1, 6-bisphosphate, 3-phospho-glycerate, isocitrate, malate and alpha-ketoglutarate had practically no effect on pyruvate kinase activity. Alanine, serine, glutamine, methionine and GMP had a weak activating effect on the enzyme. Some metabolites such as ATP, GTP, and UMP were found to be 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 synergistic effect. Inhibition by citrate was non-competitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2+. Small concentrations of the SH specific reagents, namely 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 micrograms/ml) and rose bengal (5 and 10 micrograms/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. Reagents specific for tyrosine (iodine and tetranitromethane) and tryptophan residues (N-bromosuccinimide) residues had no effect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.
Ambasht
P K
PK
Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India.
Malhotra
O P
OP
Kayastha
A M
AM
D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology*; D007887:Fabaceae / Q000201:enzymology*; D010946:Plants, Medicinal; D011770:Pyruvate Kinase / Q000737:chemistry / Q000502:physiology*
D011770:Pyruvate Kinase