Abstract
Irradiation of isocitrate lyase of castor seedling endosperm with visible light in the presence of a small concentration of Rose Bengal at pH 6.8 brings about a time-dependent exponential decay of enzyme activity. The first-order rate constant of the inactivation reaction increases with pH in the range pH 6–8 and becomes constant at higher pH values. When the enzyme is treated with diethyl pyrocarbonate (DEPC), a time-dependent exponential decay of its activity is observed and the pseudo first-order rate constant is proportional to the reagent concentration. The loss of activity is accompanied by a parallel increase in absorbance at 240 nm. The latter corresponds to modification of two histidine residues per monomeric subunit of the enzyme for complete inactivation. An analysis of the kinetic data shows that only one of these histidine residues is involved in the catalytic activity. Control experiments ruled out the involvement of residues other than histidine in inactivation by photooxidation and DEPC. Succinate protects the enzyme strongly against inactivation by both procedures but glyoxylate does not. A probable mechanism of action of isocitrate lyase is proposed and discussed in the light of these and earlier results.
Published Version
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