title: Isolation and characterization of novel long-chain acyl-CoA thioesterase/carboxylesterase isoenzymes from Candida rugosa.
abstract: Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoA's to free fatty acids and CoASH, are abundant in animal cells. However, in yeast little is known about presence and function of acyl-CoA thioesterase activity. Therefore a commercial lipase preparation from the yeast Candida rugosa was investigated and found to contain high myristoyl-CoA thioesterase activity. Hydrophobic interaction chromatography separated the activity into three peaks, of which two enzymes (YTE-1 and YTE-2) were purified to apparent homogeneity with molecular masses of about 40 kDa as determined by size-exclusion chromatography and SDS-PAGE. The employed purification protocol resulted in final preparations with specific activities of about 90 micromol/mg/min with myristoyl-CoA as substrate. YTE-1 and YTE-2 showed similar kinetic properties and YTE-1 was characterized in detail. Acyl-CoA chain-length specificity showed that YTE-1 was not active on acyl-CoAs shorter than decanoyl-CoA, at the substrate concentrations tested. The best substrates were C14-C18 acyl-CoAs with Vmax values of about 150 micromol/mg/min and Km values of 15-46 microM. The enzyme was very active with lauroyl-CoA (Vmax about 400 micromol/mg/min) although the Km was high (about 325 microM). The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin. Treatment of the protein with N-glycosidase F decreased the molecular mass about 1-2 kDa, indicating the presence of carbohydrate of the high mannose type. Diisopropyl fluorophosphate (DFP) inhibited the enzyme activity efficiently and the protein was covalently labeled with [3H]DFP. p-Chloromercuribenzoic acid inhibited the thioesterase activity but did not affect carboxylesterase activity. N-terminal sequence analysis and labeling by DFP suggest that these long-chain acyl-CoA thioesterases belong to a novel group of yeast serine esterases.
authors.affiliation: Department of Medical Laboratory Sciences and Technology, Karolinska Institutet, Huddinge University Hospital, Sweden.
authors.forename: M A
authors.initials: MA
authors.lastname: Diczfalusy
authors.forename: S E
authors.initials: SE
authors.lastname: Alexson
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry; D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D013869:Thiolester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D002729:Chloromercuribenzoates; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D013439:Sulfhydryl Reagents; D004950:Esterases; C022635:serine esterase; D002265:Carboxylic Ester Hydrolases; C063418:long-chain carboxylesterase; D013869:Thiolester Hydrolases