Purification and characterization of fatty acid synthetase from Cryptococcus neoformans.
Fatty acid synthetase has been purified from Cryptococcus neoformans 450 fold to a specific activity of 3.6 units per mg protein with an overall yield of 23%. The purified enzyme contained two non-identical subunits, Mr approximately 2.1 x 10(5) and 1.8 x 10(5). Under optimum conditions, 100 mM KCl and pH 7.5, apparent K(m) values for the substrates were: Acetyl CoA, 19 microM; Malonyl CoA, 5 microM; and NADPH, 6 microM. Product inhibition patterns were determined to be: CoA, competitive versus acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, competitive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; Palmitoyl CoA, competitive versus malonyl CoA, noncompetitive versus acetyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. These 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 before demonstrated for a fatty acid synthetase. This inhibition exhibited a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6 x 10(5) and above having Ki values below 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high positive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first purification of fatty acid synthetase from a basidiomycete.
Mahmoud
Y A
YA
Department of Biochemistry, Virginia Tech, Blacksburg 24061-0308, USA.
Abu el Souod
S M
SM
Niehaus
W G
WG
D000838:Anions / Q000494:pharmacology; D001665:Binding Sites; D002569:Cerulenin / Q000494:pharmacology; D003455:Cryptococcus neoformans / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D011487:Protein Conformation; D015032:Zinc / Q000494:pharmacology
D000838:Anions; D004791:Enzyme Inhibitors; D002569:Cerulenin; D006493:Heparin; D064429:Fatty Acid Synthases; D015032:Zinc