Figure 5: Rationalizing the identical L-asparaginase kinetic properties yet differential impact of Asp binding to WoA-P121 and WoA-S121.
The complexes with Asp reveal a different conformation for WoA-P121 (closed) and WoA-S121 (open), yet the kinetics of Asn hydrolysis is identical for the two enzyme variants. (a) To understand the differential response to Asp and Asn, we modelled an Asn molecule (grey colour) into the active site of WoA-S121 based on WoA-S121 + Asp structure. Distinguishable hydrogen-bond networks for Asn (b) and Asp (c). When Asn is in the active site, strong hydrogen bonds between the substrate’s amide (proton donor) and the hydroxyl moieties of both Thr14 and Thr93 tether the loop to the closed conformation regardless if residue 121 is a proline or serine. When Asp is in the active site, the hydrogen network is broken between the ligand and Thr14, hindering loop closure. Only if proline is in position 121, it strengthens the closed-conformation of Tyr27 (see text), which in turn stabilizes Thr14 and that suffices to keep the loop in the closed state, explaining WoA-P121 + Asp structure adopting the closed conformation. When serine replaces Pro121, the major stabilizing factor for Tyr27 is lost and it can no longer compensate the lack of bonding between the ligand and Thr14, resulting in the WoA-S121 + Asp structure adopting the open conformation.
