PURIFICATION AND CHARACTERIZATION OF FATTY-ACID SYNTHETASE FROM CRYPTOCOCCUS-NEOFORMANS

Fatty acid synthetase has been purified from Cryptococcus neoformans 4 50 fold to a specific activity of 3.6 units per mg protein with an ove rall yield of 23%. The purified enzyme contained two non-identical sub units, Mr approximately 2.1 x 10(5) and 1.8 x 10(5). Under optimum con ditions, 100 mM KCl and pH 7.5, apparent Km values for the substrates were: Acetyl CoA, 19 mu M; Malonyl CoA, 5 mu M; and NADPH, 6 mu M. Pro duct inhibition patterns were determined to be: CoA, competitive versu s acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, compet itive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; P almitoyl CoA, competitive versus malonyl CoA, noncompetitive versus ac etyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. The se product inhibition patterns are consistent with the multisite ping- pong mechanism previously proposed for the avian fatty acid synthetase complex. The cryptococcal fatty acid synthetase was inhibited by the polyanionic polymers, heparin and dextran sulfate, an effect never bef ore demonstrated for a fatty acid synthetase. This inhibition exhibite d a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6 x 10(5) and above having K-i values be low 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high posit ive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first puri fication of fatty acid synthetase from a basidiomycete.