Isolation and Characterization of Novel Long-Chain Acyl-CoA Thioesterase/Carboxylesterase Isoenzymes fromCandida rugosa

Long-chain acyl-CoA thioesterases, which catalyze the cleavage of acyl-CoAʹs 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 yeastCandida rugosawas 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 μmol/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–C18acyl-CoAs withVmaxvalues of about 150 μmol/mg/min andKmvalues of 15–46 μm. The enzyme was very active with lauroyl-CoA (Vmaxabout 400 μmol/mg/min) although theKmwas high (about 325 μm). The purified enzyme was also active on short-chain nitrophenyl esters but inactive with tributyrin. Treatment of the protein withN-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.