Purification and characterization of 3-ketoacyl-acyl carrier protein synthase III from spinach. A condensing enzyme utilizing acetyl-coenzyme A to initiate fatty acid synthesis.
The 3-ketoacyl-acyl carrier protein (ACP) synthase III from spinach was purified to homogeneity by an eight-step procedure that included an ACP-affinity column. The size of the native enzyme was M(r) = 63,000 based on gel filtration, and its subunit size was M(r) = 40,500 based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that 3-ketoacyl-ACP synthase III may be a homodimer. The purified enzyme was highly specific for acetyl-CoA and malonyl-ACP. The Km for acetyl-CoA was 5 microM when assayed in the presence of 10 microM malonyl-CoA. Acetyl-, butyryl-, and hexanoyl-ACP would not substitute for acetyl-CoA as substrates. The specificity for acetyl-CoA suggested that the physiological function of 3-ketoacyl-ACP synthase is to catalyze the initial condensation reaction in fatty acid biosynthesis. The homogeneous 3-ketoacyl-ACP synthase was capable of catalyzing acetyl-CoA:ACP transacylation but at a rate about 90-fold slower than the condensation reaction with malonyl-ACP. The 3-ketoacyl-ACP synthase was inhibited 100% by 5 mM N-ethylmaleimide or 20 mM sodium arsenite.
Clough
R C
RC
Department of Botany, Miami University, Oxford, Ohio 45056.
Matthis
A L
AL
Barnum
S R
SR
Jaworski
J G
JG
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000302:isolation & purification* / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017886:Durapatite; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis*; D006882:Hydroxyapatites; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013379:Substrate Specificity
D002352:Carrier Proteins; D005227:Fatty Acids; D006882:Hydroxyapatites; D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D017886:Durapatite; C077402:3-ketoacyl-acyl carrier protein synthase III; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase