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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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Dependence of ligand-OASS interactions on ionic strength-Fluorescence quenching titrations of ligands in the binding buffer with indicated [NaCl]. Solid line represents the fit derived from two identical site model; (A) Titrations of Cysteine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 50 mM; (open circle) 100 mM; (open diamond) 200 mM; (closed triangle) 500 mM; (B) Titrations of methionine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 100 mM; (open circle) 200 mM; (closed triangle) 500 mM; (C) Salt dependence of cysteine (closed circle) and methionine (open circle) binding constants. δlog(Kobs)/δlog[NaCl] ~ 0.8 for methionine binding.
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Figure 6: Dependence of ligand-OASS interactions on ionic strength-Fluorescence quenching titrations of ligands in the binding buffer with indicated [NaCl]. Solid line represents the fit derived from two identical site model; (A) Titrations of Cysteine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 50 mM; (open circle) 100 mM; (open diamond) 200 mM; (closed triangle) 500 mM; (B) Titrations of methionine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 100 mM; (open circle) 200 mM; (closed triangle) 500 mM; (C) Salt dependence of cysteine (closed circle) and methionine (open circle) binding constants. δlog(Kobs)/δlog[NaCl] ~ 0.8 for methionine binding.

Mentions: We have examined the interaction of methionine and cysteine with OASS under different solution conditions. Predominantly entropy driven methionine binding suggests that substrate binding may follow classical hydrophobic interactions with minimal contributions from other forces. On contrary, analyses of structure of OASS-methionine complex shows a number of polar interactions between the ligand's charged as well as polar groups and protein side chains suggesting the role of non-hydrophobic forces involved in methionine binding [15]. To understand the dependency of cysteine and methionine binding on electrolyte concentrations, we screened the complex formation in buffers with different ionic strength. Both the methionine and cysteine binding were examined by monitoring the binding processes as a function of [NaCl] (Figure 6A &6B). Binding of cysteine to StOASS showed that binding constants are not very sensitive to [NaCl] (dlogK/dlog [NaCl] ~ -0.21 ± 0.1) over the range of [NaCl] examined (0.02-0.5 M) (table 2). Interestingly, binding of methionine is more [NaCl] dependent than cysteine binding. Binding constant decreases as [NaCl] increased with dlogK/dlog [NaCl] value of ~ -0.8 ± 0.1, which indicates that methionine binding to StOASS is accompanied by the release of one anion or cation (Figure 6C). Comparison of binding constants of cysteine-StOASS and methionine-StOASS complex formation indicates that cysteine has at least 10 fold higher affinity for StOASS than methionine at higher [NaCl] (0.2-0.5 M).


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)

Dependence of ligand-OASS interactions on ionic strength-Fluorescence quenching titrations of ligands in the binding buffer with indicated [NaCl]. Solid line represents the fit derived from two identical site model; (A) Titrations of Cysteine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 50 mM; (open circle) 100 mM; (open diamond) 200 mM; (closed triangle) 500 mM; (B) Titrations of methionine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 100 mM; (open circle) 200 mM; (closed triangle) 500 mM; (C) Salt dependence of cysteine (closed circle) and methionine (open circle) binding constants. δlog(Kobs)/δlog[NaCl] ~ 0.8 for methionine binding.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Dependence of ligand-OASS interactions on ionic strength-Fluorescence quenching titrations of ligands in the binding buffer with indicated [NaCl]. Solid line represents the fit derived from two identical site model; (A) Titrations of Cysteine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 50 mM; (open circle) 100 mM; (open diamond) 200 mM; (closed triangle) 500 mM; (B) Titrations of methionine with StOASS at indicated [NaCl] in the binding buffer; (closed square) 100 mM; (open circle) 200 mM; (closed triangle) 500 mM; (C) Salt dependence of cysteine (closed circle) and methionine (open circle) binding constants. δlog(Kobs)/δlog[NaCl] ~ 0.8 for methionine binding.
Mentions: We have examined the interaction of methionine and cysteine with OASS under different solution conditions. Predominantly entropy driven methionine binding suggests that substrate binding may follow classical hydrophobic interactions with minimal contributions from other forces. On contrary, analyses of structure of OASS-methionine complex shows a number of polar interactions between the ligand's charged as well as polar groups and protein side chains suggesting the role of non-hydrophobic forces involved in methionine binding [15]. To understand the dependency of cysteine and methionine binding on electrolyte concentrations, we screened the complex formation in buffers with different ionic strength. Both the methionine and cysteine binding were examined by monitoring the binding processes as a function of [NaCl] (Figure 6A &6B). Binding of cysteine to StOASS showed that binding constants are not very sensitive to [NaCl] (dlogK/dlog [NaCl] ~ -0.21 ± 0.1) over the range of [NaCl] examined (0.02-0.5 M) (table 2). Interestingly, binding of methionine is more [NaCl] dependent than cysteine binding. Binding constant decreases as [NaCl] increased with dlogK/dlog [NaCl] value of ~ -0.8 ± 0.1, which indicates that methionine binding to StOASS is accompanied by the release of one anion or cation (Figure 6C). Comparison of binding constants of cysteine-StOASS and methionine-StOASS complex formation indicates that cysteine has at least 10 fold higher affinity for StOASS than methionine at higher [NaCl] (0.2-0.5 M).

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