The multifunctional polypeptide chains of rabbit-mammary fatty-acid synthase. Stoichiometry of active sites and active-site mapping using limited proteolysis.
Several methods have been used to label active centres on the multifunctional polypeptide chains of rabbit mammary fatty acid synthase. Experiments using [14C]acetyl-CoA or [14C]malonyl-CoA have shown that there is a single non-thiol site which binds either acetyl or malonyl groups, present at a stoichiometry of two per enzyme dimer, and representing an intermediate in the acyl transferase reaction. This adds further support to the view that the two subunits are identical and that each polypeptide chain contains up to seven active centres. However, two novel and independent methods for the quantification of the pantetheine thiol demonstrate that this prosthetic group can be present in sub-stoichiometric amounts. By studying intermediates during limited elastase digestion of fatty acid synthase labelled in different active centres, we have been able to map the positions of four active centres within the polypeptide chain. The thioesterase domain is present in a terminal location on both polypeptide chains as previously reported. The acyl carrier domain (pantetheine thiol) is located in a region of molecular weight 9000 immediately adjacent to the thioesterase domain. The acyl transferase (acyl-O-ester site) and the 3-oxoacylsynthase thiol are located in a region of molecular weight 110000 at the opposite end of the polypeptide chain to the thioesterase domain. The relationship between the disposition of the activities on the multifunctional polypeptide chains of yeast and mammalian fatty acid synthase is discussed.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D064429:Fatty Acid Synthases / Q000032:analysis*; D005260:Female; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000201:enzymology*; D010196:Pancreatic Elastase; D010204:Pantetheine / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D010204:Pantetheine; D008316:Malonyl Coenzyme A; D000105:Acetyl Coenzyme A; D064429:Fatty Acid Synthases; D010196:Pancreatic Elastase