REGULATORY PROPERTIES AND ACTIVE-SITE GROUPS OF CYTOSOLIC MUNG BEAN PYRUVATE-KINASE

Properties of mung bean pyruvate kinase were studied and the active si te groups were derived. Metabolites like AMP, glucose, glucose-6-phosp hate, fructose-6-phosphate, fructose-1,6-bisphosphate, 3-phospho-glyce rate, isocitrate, malate and alpha-ketoglutarate had practically no ef fect on pyruvate kinase activity. Alanine, serine, glutamine, methioni ne and GMP had a weak activating effect on the enzyme. Some metabolite s such as ATP, GTP, and UMP were found to be weakly inhibitory. Modera te to strong inhibition was observed with citrate, succinate, glutamat e and oxalate. Inhibition brought about by ATP and citrate when presen t together showed synergistic effect. Inhibition by citrate was non-co mpetitive with respect to both PEP and ADP suggesting the presence of a regulatory site. Mung bean pyruvate kinase showed half optimal activ ity at pH 6.6 and 8.9 at saturating concentrations of PEP, ADP and Mg2 +. Small concentrations of the SH specific reagents, namely iodoacetam ide (0.1 and 0.2 mM), N-ethylmaleimide(0.05-0.1 mM) and p-chloromercur ibenzoate (0.1 mM) inactivated the enzyme; single exponential loss of activity was observed in each case. Photooxidation of the enzyme in th e presence of methylene blue (100 and 200 mu g/ml) and rose bengal (5 and 10 mu g/ml) also led to a single exponential activity decay. When the enzyme was treated with diethyl pyrocarbonate(DEP), a time depende nt exponential decay in its activity was observed with a parallel incr ease in absorbance at 240 nm. PEP protected the enzyme against inactiv ation by DEP. Reagents specific for tyrosine (iodine and tetranitromet hane) and tryptophan residues (N-bromosuccinimide) residues had no eff ect. These observations confirm that SH and imidazole groups are vital for the activity of the enzyme.