Modification of the active site of isocitrate lyase from watermelon cotyledons

Abstract

Isocitrate lyase (EC 4.1.3.1) from watermelon cotyledons was modified by diethylpyrocarbonate and by the affinity labels 3-bromopyruvate and itaconate epoxide. The reaction with diethylpyrocarbonate, carried out at 30 °C in sodium phosphate, pH 6.5, modified (per subunit) 5 histidines in the absence and 4 in the presence of substrate. The kinetics were nonsaturating with respect to diethylpyrocarbonate and the enzyme was protected against modification by substrate or both products together. Hydroxylamine (0.5M ) reversed both histidine modification and inactivation. The reaction with 3-bromopyruvate, carried out at 30 °C in 4-morpholinepropanesulfonic acid, pH 7.7, modified (per subunit) 1 sulfhydryl in the absence and 0 in the presence of substrate. The reaction showed saturation kinetics (K_BrP = 1.4 × 10^-5 m) and D_s-isocitrate offers competitive protection (K_I = 0.2-0.3 mM; K_m = 0.25 mM). The reaction with itaconate epoxide, carried out at 30 °C in sodium phosphate, pH 7.0, was also saturating (K_ItEp = 16.4 mM) and the reversible inhibitor, itaconate (K_I = 50 µm; K_i = 22.5 µm) as well as the product, succinate (K_s = 10.4 mM; K_i = 4.5 mM) offer competitive protection. Hydroxylamine (l_M , pH 7.0) reversed inactivation of the enzyme, indicating modification of a carboxylate residue at the active site. In summary, three different amino acid residues have been modified in the active site domain of watermelon isocitrate lyase.