Pancreatic cholesterol esterases. 2. Purification and characterization of human pancreatic fatty acid ethyl ester synthase

Abstract

Human pancreatic fatty acid ethyl ester synthase has been isolated and purified 1200-fold to homogeneity, and its activities, binding properties, and N-terminal amino acid sequence indicate that it is a member of the lipase family. This 52-kDa monomeric protein is present at 0.6-1.2 mg/g of pancreas, and it catalyzes the synthesis and hydrolysis of ethyl oleate at rates of 2400 nmol mg-1 h-1 and 30 nmol mg-1 h-1, respectively. Kinetic analyses reveal a pronounced substrate specificity for unsaturated octadecanoic fatty acids, with ethyl ester synthetic rates of 2400 nmol mg-1 h-1 (linoleic), 2400 nmol mg-1 h-1 (oleic), 400 nmol mg-1 h-1 (arachidonic), 300 nmol mg-1 h-1 (palmitic), and 100 nmol mg-1 h-1 (stearic). Like cholesterol esterase, the enzyme binds to immobilized heparin, and this property was critical for its purification to homogeneity. Its N-terminal amino acid sequence is virtually identical with that reported for human triglyceride lipase, NH2-X-Glu-Val-Cys-5Tyr-Glu-Arg-Leu-Gly-10Cys-Phe-Ser-Asp- Asp-15Ser-Pro-Trp-Ser-Gly-20Ile, and it differs by only four residues from that reported for porcine pancreatic lipase. The synthase purified here also cleaves triglycerides, hydrolyzing triolein at a rate of 30 nmol mg-1 h-1, and this activity is stimulated by colipase and inhibited by sodium chloride. Conversely, commercially available porcine triglyceride lipase exhibits fatty acid ethyl ester synthase activity (1530 nmol mg-1 h-1) and hydrolyzes triolein at a rate of 23 nmol mg-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

“…20 This activity is noted in pancreatic homogenates in the presence of ethanol and after ethanol infusion 51,52 and may exceed the lipolytic capacity of the putative enzyme by >60-fold. 25 This suggests that in the presence of sufficient ethanol, FAs may be converted to FAEEs in vivo. Our current study clearly shows that FAEEs are less toxic than their parent UFAs at equivalent concentrations.…”

“…21 The putative enzyme responsible for FAEE generation has remained elusive. The enzyme was mostly referred to as carboxylesterase 25 or cholesterol esterase in the 1990s. 25 It was later proposed that formation of FAEEs is catalyzed by both an acyl-CoA:ethanol O-acyltransferase and a FAEE synthase which use FAs and acyl-CoA as substrates, respectively.…”

“…The enzyme was mostly referred to as carboxylesterase 25 or cholesterol esterase in the 1990s. 25 It was later proposed that formation of FAEEs is catalyzed by both an acyl-CoA:ethanol O-acyltransferase and a FAEE synthase which use FAs and acyl-CoA as substrates, respectively. 65 Subsequently, this enzyme that has its first 15 amino acids identical to pancreatic cholesterol esterase 66 was noted to be sensitive to inhibitors of serine proteases.…”

“…11,12 Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,20,23 and the putative enzyme for this was thought to be a lipase. 24,25 It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold. 25 Triglyceride (TG) forms >80% of the adipocyte mass, 26e28 oleic acid (OA) being the most enriched FA.…”

“…24,25 It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold. 25 Triglyceride (TG) forms >80% of the adipocyte mass, 26e28 oleic acid (OA) being the most enriched FA. 9,29 We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.…”