Non-lethal disease variants of essential enzymes must impair, but not abolish, function by altering the protein’s underlying conformational landscape. However, the vast majority of enzyme disease variants remain uncharacterized and many more variants have unknown significance. Here, as part of an undergraduate course based research experience (CURE), we structurally and functionally characterized four pathogenic variants of human glutamine synthetase (GS) (R324C, R324S, R341C, and R341H) using cryo-EM, differential scanning fluorimetry, and steady-state kinetics. All variants displayed similar baseline thermodynamic stability compared to wild type. However, while wild type was strongly stabilized by binding a transition state analog (methionine sulfoximine + ATP), none of the variants exhibited this stabilization. Accordingly, all variants showed defects in Michaelis-Menten complex formation, with KM increases up to 100-fold, while kcat remained largely unchanged. Cryo-EM structures captured under apo-, transition state, and turnover conditions (2.1–2.7 Å resolution) revealed that variants could still form productive transition state complexes with overall conformations similar to wild type, but with localized remodeling in the active site. Under turnover, ADP density was observed in both wild type and variants, consistent with product release being rate-limiting—a conclusion further supported by kinetic solvent viscosity analyses. On closer inspection, R324C and R324S disrupted Michaelis-Menten complex formation by altering a Mg(II)-coordination site, while the non-active site variant R341C introduced additional flexibility into helix H8 and the substrate-gating loop 295-311 (containing catalytic residue E305), explaining its more severe kinetic phenotype. Together, these results show how pathogenic GS variants remodel conformational space to impair Michaelis-Menten complex formation while leaving global structure and catalytic turnover intact, providing insight into the conformational control of enzyme function.
Mejia et al. (Sun,) studied this question.