title: Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines.
abstract: Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
authors.forename: Oksana
authors.initials: O
authors.lastname: Lockridge
authors.forename: Weihua
authors.initials: W
authors.lastname: Xue
authors.forename: Andrea
authors.initials: A
authors.lastname: Gaydess
authors.forename: Hasmik
authors.initials: H
authors.lastname: Grigoryan
authors.forename: Shi-Jian
authors.initials: SJ
authors.lastname: Ding
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Schopfer
authors.forename: Steven H
authors.initials: SH
authors.lastname: Hinrichs
authors.forename: Patrick
authors.initials: P
authors.lastname: Masson
mesh_terms: D000107:Acetylation; D001665:Binding Sites; D004950:Esterases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D010206:Papain; D012709:Serum Albumin / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
chemical_list: D012709:Serum Albumin; D014443:Tyrosine; D004950:Esterases; D010206:Papain; D008239:Lysine