Modification of the active site of isocitrate lyase from watermelon cotyledons.
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 degrees 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 (0.5 M) reversed both histidine modification and inactivation. The reaction with 3-bromopyruvate, carried out at 30 degrees 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 (KBrP = 1.4 X 10(-5)M) and Ds-isocitrate offers competitive protection (KI = 0.2-0.3 mM; Km = 0.25 mM). The reaction with itaconate epoxide, carried out at 30 degrees C in sodium phosphate, pH 7.0, was also saturating (KItEp = 16.4 mM) and the reversible inhibitor, itaconate (KI = 50 microM; Ki = 22.5 microM) as well as the product, succinate (KS = 10.4 mM; Ki = 4.5 mM) offer competitive protection. Hydroxylamine (1 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.
D000345:Affinity Labels / Q000138:chemical synthesis; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004047:Diethyl Pyrocarbonate; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011773:Pyruvates; D013386:Succinates / Q000138:chemical synthesis
D000345:Affinity Labels; D011773:Pyruvates; D013386:Succinates; C017092:bromopyruvate; C044122:itaconate epoxide; D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase; D004047:Diethyl Pyrocarbonate