Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines.
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.
Lockridge
Oksana
O
Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Xue
Weihua
W
Gaydess
Andrea
A
Grigoryan
Hasmik
H
Ding
Shi-Jian
SJ
Schopfer
Lawrence M
LM
Hinrichs
Steven H
SH
Masson
Patrick
P
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
D012709:Serum Albumin; D014443:Tyrosine; D004950:Esterases; D010206:Papain; D008239:Lysine