Isocitrate Lyase from Flax

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Abstract
The cleavage of Ds-isocitrate catalyzed by isocitrate lyase from L. usitatissimum results in the ordered release of succinate and glyoxylate. The glyoxylate analog 3-bromopyruvate irreversibly inactivates the flax enzyme in a process exhibiting saturation kinetics and protection by glyoxylate or isocitrate or the competitive inhibitor L-tartrate. Succinate provides considerably less protection. Results with 3-bromopyruvate suggest that this reagent modifies plant and prokaryotic isocitrate lyases differently. Treatment of the tetrameric 264,000-dalton flax enzyme with carboxypeptidase A results in a release of 1 histidine/subunit which is concordant with loss of activity. The only N-terminal residue is methionine. Treatment of flax enzyme with diethylpyrocarbonate at pH 6.5 selectively modifies 2 histidines per 67,000-dalton subunit. The reaction of 1 histidine residue is abolished by the binding of L-tartrate and the modification of 1 is coincident with inactivation. The carboxy-terminal and active-site modifications establish that 1 histidine residue/monomer is essential in the flax enzyme and considerably extend information heretofore available only for fungal and bacterial isocitrate lyase.