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Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin.

Ahmad E, Rabbani G, Zaidi N, Ahmad B, Khan RH - PLoS ONE (2012)

Bottom Line: These findings were compared to HSA-hydrolase activity.We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators.We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.

ABSTRACT
Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

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Enzyme kinetics for HSA.(A) is the Michaeil-Menton equation based; (B) is the Lineweaver-Burk plots of reaction velocity versus substrate concentration for enzyme kinetics of HSA in absence and presence of pollutants (1∶5); (C) is the plot of % activity against pollutant concentrations at a fixed substrate and HSA concentration.
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pone-0038372-g008: Enzyme kinetics for HSA.(A) is the Michaeil-Menton equation based; (B) is the Lineweaver-Burk plots of reaction velocity versus substrate concentration for enzyme kinetics of HSA in absence and presence of pollutants (1∶5); (C) is the plot of % activity against pollutant concentrations at a fixed substrate and HSA concentration.

Mentions: Figure 8A shows the pattern of nitrocefin hydrolysis by HSA in the absence and presence of a fixed concentration of these three pollutant molecules according to Michaelis-Menten equation. The rectangular hyperbolic curves are the characteristic feature of a true enzyme. Further, the reciprocal of substrate concentration against their respective reciprocal of degradation rate were plotted on the basis of Lineweaver-Burk equation (Figure 8B). The obtained values for all the extrinsic (Vmax) and intrinsic (Km, kcat) properties of an enzyme are listed in Table 5. In presence of pollutants the decrease in Km from 1.05×10−4 M to 2.86×10−5 M reveals the increase in affinity and tighter substrate binding by the albumin. The turnover number (kcat) of the albumin complexed with pollutants is also increased up to ∼10 fold. The second order rate constant kcat/Km indicates the catalytic efficiency and kinetic perfection of the enzyme in transforming substrates. The higher the kcat/Km is, the better the enzyme works on that substrate. A comparison of kcat/Km for the same enzyme with substrates in different conditions is widely used as a measure of enzyme effectiveness. The kcat/Km value increased from 6.57×106 to 3.71×107 M−1 min-1 in the absence and presence of pollutants (Table 5). The pollutant induced catalytic activation of HSA allows the reaction to approach the limit of maximum diffusion just like in an ideal enzyme (acetylcholinesterase) where every interaction with substrate yields a product and for these enzymes, from the diffusion theory, the value of kcat/Km ranges 6×109–6×1010 M−1 min−1[30]. The results of enzyme kinetics suggest that the pollutants act as activators. For the determination of activator category, whether it is essential or non-essential activation, the enzyme activity of HSA in presence of different concentrations of pollutant molecules is shown in Figure 8C and the obtained values are given in Table 5. Here, the non-linear enhancement in activity is observed. This shows a non-essential activation as the non-linearity is prerequisite of such type of activation. The mechanism of hydrolase activity of HSA occurs through irreversible coupling through acylation of nucleophilic group (−NH2, −OH, or −SH) of the protein with concurrent rupture of the β-lactam ring between the carbonyl carbon and nitrogen (Figure 1). At pH 7.4, the ε-amino group of lysine residues and guanidinium –NH2 group of arginine residues are positively charged. Hence these –NH2 groups are electrophilic but not nucleophilic, and cannot acylate the β-lactam ring. The –SH of Cys34 was unaffected because of the absence of any significant change in near-UV CD spectra of HSA-nitrocefin complex (data not shown) in the range between 250 nm to 255 nm. Hence the residues involved in enzymatic activity are –OH containing residues such as serine, threonine and tyrosine in the vicinity of the active site. A comparison of free and pollutant bound HSA reveals that a random coil of the protein molecule rotates by some angle, resulting in movements of the polypeptide chain and in closing the cleft where nitrocefin is bound (Figure 9). The above results point to a large conformational change consistent with the hinge motion of domains observed in other proteins. In the present study, the change in hydrodynamic radii upon ligand binding calculated from the DLS data is the same as that observed by other groups in different protein-ligand models from different technical approaches, validated by domain movements in solution. Hence, we can also say that pollutants induce domain movement in HSA.


Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin.

Ahmad E, Rabbani G, Zaidi N, Ahmad B, Khan RH - PLoS ONE (2012)

Enzyme kinetics for HSA.(A) is the Michaeil-Menton equation based; (B) is the Lineweaver-Burk plots of reaction velocity versus substrate concentration for enzyme kinetics of HSA in absence and presence of pollutants (1∶5); (C) is the plot of % activity against pollutant concentrations at a fixed substrate and HSA concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369883&req=5

pone-0038372-g008: Enzyme kinetics for HSA.(A) is the Michaeil-Menton equation based; (B) is the Lineweaver-Burk plots of reaction velocity versus substrate concentration for enzyme kinetics of HSA in absence and presence of pollutants (1∶5); (C) is the plot of % activity against pollutant concentrations at a fixed substrate and HSA concentration.
Mentions: Figure 8A shows the pattern of nitrocefin hydrolysis by HSA in the absence and presence of a fixed concentration of these three pollutant molecules according to Michaelis-Menten equation. The rectangular hyperbolic curves are the characteristic feature of a true enzyme. Further, the reciprocal of substrate concentration against their respective reciprocal of degradation rate were plotted on the basis of Lineweaver-Burk equation (Figure 8B). The obtained values for all the extrinsic (Vmax) and intrinsic (Km, kcat) properties of an enzyme are listed in Table 5. In presence of pollutants the decrease in Km from 1.05×10−4 M to 2.86×10−5 M reveals the increase in affinity and tighter substrate binding by the albumin. The turnover number (kcat) of the albumin complexed with pollutants is also increased up to ∼10 fold. The second order rate constant kcat/Km indicates the catalytic efficiency and kinetic perfection of the enzyme in transforming substrates. The higher the kcat/Km is, the better the enzyme works on that substrate. A comparison of kcat/Km for the same enzyme with substrates in different conditions is widely used as a measure of enzyme effectiveness. The kcat/Km value increased from 6.57×106 to 3.71×107 M−1 min-1 in the absence and presence of pollutants (Table 5). The pollutant induced catalytic activation of HSA allows the reaction to approach the limit of maximum diffusion just like in an ideal enzyme (acetylcholinesterase) where every interaction with substrate yields a product and for these enzymes, from the diffusion theory, the value of kcat/Km ranges 6×109–6×1010 M−1 min−1[30]. The results of enzyme kinetics suggest that the pollutants act as activators. For the determination of activator category, whether it is essential or non-essential activation, the enzyme activity of HSA in presence of different concentrations of pollutant molecules is shown in Figure 8C and the obtained values are given in Table 5. Here, the non-linear enhancement in activity is observed. This shows a non-essential activation as the non-linearity is prerequisite of such type of activation. The mechanism of hydrolase activity of HSA occurs through irreversible coupling through acylation of nucleophilic group (−NH2, −OH, or −SH) of the protein with concurrent rupture of the β-lactam ring between the carbonyl carbon and nitrogen (Figure 1). At pH 7.4, the ε-amino group of lysine residues and guanidinium –NH2 group of arginine residues are positively charged. Hence these –NH2 groups are electrophilic but not nucleophilic, and cannot acylate the β-lactam ring. The –SH of Cys34 was unaffected because of the absence of any significant change in near-UV CD spectra of HSA-nitrocefin complex (data not shown) in the range between 250 nm to 255 nm. Hence the residues involved in enzymatic activity are –OH containing residues such as serine, threonine and tyrosine in the vicinity of the active site. A comparison of free and pollutant bound HSA reveals that a random coil of the protein molecule rotates by some angle, resulting in movements of the polypeptide chain and in closing the cleft where nitrocefin is bound (Figure 9). The above results point to a large conformational change consistent with the hinge motion of domains observed in other proteins. In the present study, the change in hydrodynamic radii upon ligand binding calculated from the DLS data is the same as that observed by other groups in different protein-ligand models from different technical approaches, validated by domain movements in solution. Hence, we can also say that pollutants induce domain movement in HSA.

Bottom Line: These findings were compared to HSA-hydrolase activity.We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators.We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.

ABSTRACT
Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

Show MeSH
Related in: MedlinePlus