Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit. Reevaluation of the side-by-side, antiparallel subunit model.
The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.
Witkowski
A
A
Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Joshi
A K
AK
Rangan
V S
VS
Falick
A M
AM
Witkowska
H E
HE
Smith
S
S
D000096:Acetone / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008970:Molecular Weight; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D000096:Acetone; D010204:Pantetheine; C047048:1,3-dibromo-2-propanone; D064429:Fatty Acid Synthases; D003545:Cysteine; C003129:4'-phosphopantetheine