PURIFICATION AND PROPERTIES OF ISOCITRATE LYASE FROM ASPERGILLUS-NIDULANS, A MODEL ENZYME TO STUDY CATABOLITE INACTIVATION IN FILAMENTOUS FUNGI

In order to facilitate the purification of isocitrate lyase from Asper gillus nidulans, the isocitrate lyase overexpressing strain JCB4a was derived. Isocitrate lyase was purified to homogeneity by the criterion of polyacrylamide gel electrophoresis and anti-isocitrate lyase polyc lonal antibodies were raised. Stabilization of purified enzyme, when s tored at -20 degrees C, required the addition of 1 mM dithiothreitol ( DTT) plus I mM ethylenediaminetetraacetate (EDTA). Aspergillus nidulan s isocitrate lyase is a multimeric enzyme wi th a native molecular wei ght of 240 kDa and composed of four monomers of 59 kDa. The enzyme req uired 5 mM Mg2+ and 1 mM DTT or cysteine for full activity. EDTA at I mM replaced the requirement of a thiol compound for activity. The K-m for threo-D-S-isocitrate was 0.050 mM, and the enzyme activity was inh ibited by succinate, itaconate and structural analogs of glyoxylate as well as by fructose-1,6-bisphosphate.