Purification and properties of isocitrate lyase from Aspergillus nidulans, a model enzyme to study catabolite inactivation in filamentous fungi

Abstract

In order to facilitate the purification of isocitrate lyase from Aspergillus 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 polyclonal antibodies were raised. Stabilization of purified enzyme, when stored at – 20 °C, required the addition of 1 mM dithiothreitol (DTT) plus 1 mM ethylenediaminetetraacetate (EDTA). Aspergillus nidulans isocitrate lyase is a multimeric enzyme with a native molecular weight of 240 kDa and composed of four monomers of 59 kDa. The enzyme required 5 mM Mg and 1 mM DTT or cysteine for full activity. EDTA at 1 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 inhibited by succinate, itaconate and structural analogs of glyoxylate as well as by fructose-1,6-bisphosphate.