Molecular characterization of human eye and heart fatty acid ethyl ester synthase/carboxylesterase by site-directed mutagenesis.
The FAEES/carboxylesterase enzyme nonoxidatively metabolizes alcohol into FAEEs, potentially toxic molecules linked with alcohol-induced end-organ damage. Our laboratory has isolated and characterized a FAEES/carboxylesterase cDNA from human eye and heart (G12). In an effort to further characterize the G12 enzyme, this laboratory has analyzed the FAEES activity, as well as performed mutagenic studies on a proposed active site of G12. We have mutated Ser 204 and His 451 in the G12 enzyme in an attempt to characterize the active site. We found that both mutations cause almost total loss of carboxylesterase enzyme activity with the native enzyme remaining active for carboxylesterase activity (560 nmol/mg protein/h for G12). With oleic acid as a substrate, the FAEES activity was 170 nmol/mg protein/h for G12, 10.8 nmol/mg protein/h for M204, and 8.5 nmol/mg protein/h for M451. When ethanol was used as substrate, the FAEES activity was 240 nmol/mg protein/h for G12, 15.1 nmol/mg protein/h for M204, and 6.2 nmol/mg protein/h for M451. Since both carboxylesterase and FAEES enzyme activities are significantly lowered by mutating Ser 204 and His 451 of the G12 enzyme, this study indicates that these residues may be important for the key enzymatic activities of the enzyme.
Bora
Puran S
PS
Department of Internal Medicine, Division of Cardiology and Geriatric Medicine, Saint Louis University Medical Center, St. Louis, MO 63110, USA. psbora01@louisville.edu
Guruge
Bandula L
BL
Bora
Nalini S
NS
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D000431:Ethanol / Q000737:chemistry*; D005123:Eye / Q000737:chemistry* / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009206:Myocardium / Q000737:chemistry* / Q000201:enzymology; D051381:Rats; D017385:Sequence Homology; D013329:Structure-Activity Relationship
D000431:Ethanol; D000217:Acyltransferases; D002265:Carboxylic Ester Hydrolases; C043403:fatty acyl ethyl ester synthase