Purification and properties of 2-methylcitrate dehydratase from Yarrowia lipolytica.

Abstract

2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase) functioning at the methylcitric acid cycle of propionyl-CoA oxidation was purified from a cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. Disc gel electrophoresis proved that the enzyme preparation was homogeneous. The molecular weight was about 79,000 in determinations by gel filtration and SDS-disc electrophoresis. The enzyme was composed of 685 residues of amino acid per molecule. The enzyme showed an isoelectric point of 3.9. No cofactor was required for full enzyme activity. The enzyme was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate, but not by any chelating reagents. The enzyme competitively inhibited by citrate (Ki=4.5×10−4M), threo-ds-isocitrate (Ki=1.2×10−2M), threo-ds-2-methylisocitrate (Ki=1.1×10−3M), tricarballylate (Ki= 3.7×10−2M), and DL-fluorocitrate (Ki=9.5×10−4M).