The carboxyl-terminal region of thioesterase II participates in the interaction with fatty acid synthase. Use of electrospray ionization mass spectrometry to identify a carboxyl-terminally truncated form of the enzyme.
Medium-chain S-acyl fatty acid synthase thioester hydrolase (thioesterase II), a discrete 263-residue serine active-site enzyme, modifies the product specificity of the de novo lipogenic pathway in certain specialized tissues by hydrolyzing the thioester bond linking the growing acyl chain to the 4'-phosphopantetheine of the fatty acid synthase. Modification of one thioesterase II cysteine thiol with thionitrobenzoate inhibited interaction with the S-acyl-fatty acid synthase substrate but not with acyl-CoA model substrates. The identity of the sensitive cysteine residue was determined by treatment of the thionitrobenzoyl enzyme with cyanide and cleavage at the amino-terminal side of the S-cyanocysteinyl residue. Two small cleavage products were isolated; their molecular masses (889 and 675 Da) and amino acid compositions indicated that both originated from cleavage at Cys256. A new technique of electrospray ionization mass spectrometry was utilized to confirm that the heterogeneity displayed by the products of S-cyanocysteinyl cleavage resulted from the presence, in the purified preparations, of both full-length and a truncated form of the enzyme missing the carboxyl-terminal Leu-Thr peptide. The proportion of full-length polypeptide present appeared to correlate with the activity of the enzyme toward its natural substrate. The results of modification of Cys256 by thionitrobenzoate and removal of residues 262 and 263 by endogenous proteases indicate that integrity of the carboxyl-terminal region is important for interaction with its acyl-fatty acid synthase substrate.
Witkowska
H E
HE
Children's Hospital Oakland Research Institute, California 94609.
Green
B N
BN
Smith
S
S
D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001881:Borates; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005260:Female; D008321:Mammary Glands, Animal / Q000201:enzymology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011190:Potassium Cyanide / Q000494:pharmacology; D051381:Rats; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000214:Acyl Coenzyme A; D001881:Borates; D010446:Peptide Fragments; C010634:sodium borate; D004228:Dithionitrobenzoic Acid; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D003545:Cysteine; D011190:Potassium Cyanide