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Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes.

Banerjee S, Ekka MK, Kumaran S - BMC Biochem. (2011)

Bottom Line: Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined.Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh, India.

ABSTRACT

Background: The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine).

Results: Ligand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (ΔH(St-Met) = -1.5 ± 0.1 kJ/mol; TΔS(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.

Conclusions: We show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

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ITC analysis of the interaction between substrate, OAS (O-acetylserine) and OASS. Data is plotted as heat signal (μJ/sec) versus time (min) in the upper panel in each fig. Lower panel-integrated heat responses per injection from panel A plotted as normalized heat per mole of injectant. The solid line represents the best fit of the data to a two independent site binding model (eqn 2). (A) Titration of StOASS (4.5 μM) with OAS (5 mM) at 25°C. (B) Titration of HiOASS (4.5 μM) with OAS (5 mM) at 25°C. Both titrations were performed in the same buffer (20 mM Tris, pH 8.0, 50 mM NaCl).
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Figure 3: ITC analysis of the interaction between substrate, OAS (O-acetylserine) and OASS. Data is plotted as heat signal (μJ/sec) versus time (min) in the upper panel in each fig. Lower panel-integrated heat responses per injection from panel A plotted as normalized heat per mole of injectant. The solid line represents the best fit of the data to a two independent site binding model (eqn 2). (A) Titration of StOASS (4.5 μM) with OAS (5 mM) at 25°C. (B) Titration of HiOASS (4.5 μM) with OAS (5 mM) at 25°C. Both titrations were performed in the same buffer (20 mM Tris, pH 8.0, 50 mM NaCl).

Mentions: To the best of our knowledge, ITC has not been used to study ligand binding properties of OASS for quantifying the interaction parameters for OASS-ligand complex formation. The energetics of OASS binding to OAS, cysteine, methionine, serine, and isoleucine were examined. We performed isothermal titration calorimetry experiments at 25°C to probe the thermodynamics of OAS binding to OASS. Two titrations performed by injecting OAS to solution containing StOASS and HiOASS are shown in Figure 3A and 3B. Heat signals were integrated, and the binding isotherm was analyzed using two independent site model. Two independent titrations show that binding of OAS is an exothermic reaction and binding isotherms can be fitted to the same model. The analysis of OAS binding to StOASS yielded an enthalpy value of 0.43 ± 0.2 kJ/M and an apparent binding constant of ~ 1.8 ± 0.6 × 103 M-1 (ΔG = -17.6 kJ/M; Kd = 0.56 mM). Similarly, analysis of interactions of other two OASS with OAS yielded enthalpy values of 0.45 ± 0.3 kJ/M (HiOASS) and 0.53 ± 0.3 kJ/M (MtOASS). The binding constants obtained for both HiOASS and MtOASS were similar (~ 1.3 ± 0.4 × 103 M-1; ΔG = -16.9 kJ/M; Kd ~ 0.77 mM for HiOASS). In all three cases, the enthalpy contributes less than 3% to the total binding free energy. This is surprising because OAS is a small metabolite and has higher degrees of freedom in solution as compared to the bound form. Therefore, the entropy change upon complex formation is expected to be unfavorable for OAS-enzyme interaction. On the contrary, OAS interaction which includes its initial reaction and subsequent binding is predominantly driven by favorable entropy component in both cases.


Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes.

Banerjee S, Ekka MK, Kumaran S - BMC Biochem. (2011)

ITC analysis of the interaction between substrate, OAS (O-acetylserine) and OASS. Data is plotted as heat signal (μJ/sec) versus time (min) in the upper panel in each fig. Lower panel-integrated heat responses per injection from panel A plotted as normalized heat per mole of injectant. The solid line represents the best fit of the data to a two independent site binding model (eqn 2). (A) Titration of StOASS (4.5 μM) with OAS (5 mM) at 25°C. (B) Titration of HiOASS (4.5 μM) with OAS (5 mM) at 25°C. Both titrations were performed in the same buffer (20 mM Tris, pH 8.0, 50 mM NaCl).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141655&req=5

Figure 3: ITC analysis of the interaction between substrate, OAS (O-acetylserine) and OASS. Data is plotted as heat signal (μJ/sec) versus time (min) in the upper panel in each fig. Lower panel-integrated heat responses per injection from panel A plotted as normalized heat per mole of injectant. The solid line represents the best fit of the data to a two independent site binding model (eqn 2). (A) Titration of StOASS (4.5 μM) with OAS (5 mM) at 25°C. (B) Titration of HiOASS (4.5 μM) with OAS (5 mM) at 25°C. Both titrations were performed in the same buffer (20 mM Tris, pH 8.0, 50 mM NaCl).
Mentions: To the best of our knowledge, ITC has not been used to study ligand binding properties of OASS for quantifying the interaction parameters for OASS-ligand complex formation. The energetics of OASS binding to OAS, cysteine, methionine, serine, and isoleucine were examined. We performed isothermal titration calorimetry experiments at 25°C to probe the thermodynamics of OAS binding to OASS. Two titrations performed by injecting OAS to solution containing StOASS and HiOASS are shown in Figure 3A and 3B. Heat signals were integrated, and the binding isotherm was analyzed using two independent site model. Two independent titrations show that binding of OAS is an exothermic reaction and binding isotherms can be fitted to the same model. The analysis of OAS binding to StOASS yielded an enthalpy value of 0.43 ± 0.2 kJ/M and an apparent binding constant of ~ 1.8 ± 0.6 × 103 M-1 (ΔG = -17.6 kJ/M; Kd = 0.56 mM). Similarly, analysis of interactions of other two OASS with OAS yielded enthalpy values of 0.45 ± 0.3 kJ/M (HiOASS) and 0.53 ± 0.3 kJ/M (MtOASS). The binding constants obtained for both HiOASS and MtOASS were similar (~ 1.3 ± 0.4 × 103 M-1; ΔG = -16.9 kJ/M; Kd ~ 0.77 mM for HiOASS). In all three cases, the enthalpy contributes less than 3% to the total binding free energy. This is surprising because OAS is a small metabolite and has higher degrees of freedom in solution as compared to the bound form. Therefore, the entropy change upon complex formation is expected to be unfavorable for OAS-enzyme interaction. On the contrary, OAS interaction which includes its initial reaction and subsequent binding is predominantly driven by favorable entropy component in both cases.

Bottom Line: Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined.Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh, India.

ABSTRACT

Background: The importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine).

Results: Ligand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (ΔH(St-Met) = -1.5 ± 0.1 kJ/mol; TΔS(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.

Conclusions: We show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

Show MeSH
Related in: MedlinePlus