Abstract
The hydrolysis of 4-nitrophenyl esters of hexanoate (NphOHe) and decanoate (NphODe) by human serum albumin (HSA) at Tyr411, located at the FA3-FA4 site, has been investigated between pH 5.8 and 9.5, at 22.0°C. Values of K s, k +2, and k +2/K s obtained at [HSA] ≥ 5×[NphOXx] and [NphOXx] ≥ 5×[HSA] (Xx is NphOHe or NphODe) match very well each other; moreover, the deacylation step turns out to be the rate limiting step in catalysis (i.e., k +3 << k +2). The pH dependence of the kinetic parameters for the hydrolysis of NphOHe and NphODe can be described by the acidic pK a-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate. The inhibition of the HSA-Tyr411-catalyzed hydrolysis of NphOHe, NphODe, and 4-nitrophenyl myristate (NphOMy) by five inhibitors (i.e., diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol) has been investigated at pH 7.5 and 22.0°C, resulting competitive. The affinity of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol for HSA reflects the selectivity of the FA3-FA4 cleft. Under conditions where Tyr411 is not acylated, the molar fraction of diazepam, diflunisal, ibuprofen, and 3-indoxyl-sulfate bound to HSA is higher than 0.9 whereas the molar fraction of propofol bound to HSA is ca. 0.5.
Highlights
Theenzymatic activity of human serum albumin (HSA) was first reported in 1951 and investigated extensively for decades
The pH dependence of the kinetic parameters for the hydrolysis of nitrophenyl esters of hexanoate (NphOHe) and NphODe can be described by the acidic pKa-shift of a single amino acid residue, which varies from 8.9 in the free HSA to 7.6 and 7.0 in the HSA:NphOHe and HSA:NphODe complex, respectively; the pK>a-shift appears to be correlated to the length of the fatty acid tail of the substrate
As previously reported for the HSA-Tyr411-catalyzed hydrolysis of NphOAc and NphOMy [12,13], the determination of kinetic parameters of Fig. 1 is simplified by the fact that the formation of the HSA:NphOHe and HSA:NphODe complexes can be treated as a rapid equilibrium process
Summary
The (pseudo-)enzymatic activity of human serum albumin (HSA) was first reported in 1951 and investigated extensively for decades. HSA displays esterase, RNA-hydrolyzing, enolase, glucuronidase, lipid peroxidase, aldolase glutathione-linked thiol peroxidase, and PLOS ONE | DOI:10.1371/journal.pone.0120603. Inhibition of the Esterase-Like Activity of HSA anti-oxidant activities. Heme binding confers to HSA globin-like (pseudo-)enzymatic properties, including detoxification of reactive nitrogen and oxygen species as well as catalase and peroxidase activities. HSA(-heme) (pseudo-)enzymatic properties are modulated allosterically and inhibited competitively [1,2]. The physiological importance of the esterase activity of HSA is obscure and the natural substrates are still unknown, HSA displays esterase activity towards several substrates including 4-nitrophenyl acetate (NphOAc), α-naphthyl acetate, phenyl acetate, 1-naphthyl Nmethylcarbamate, β-naphthyl acetate, aspirin, ketoprofen glucuronide, carprofen acylglucuronide, cyclophosphamide, nicotinate esters, long and short-chain FA esters (e.g., 4-nitrophenyl myristate; NphOMy), octanoyl ghrelin, organophosphorus pesticides, carbaryl, 2-nitrotrifluoroacetanilide, 2-nitroacetanilide, and nerve agents [1,2,3]
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