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.