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

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.