High Resolution Crystal Structure of the Pyruvate Kinase Tetramer in Complex with the Allosteric Activator Mitapivat/AG-348

Pyruvate kinase (PK) deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway. In recent years, Mitapivat (MTPV, AG-348) has emerged as a notable allosteric activator for treating PK deficiency. However, the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated. To shed light on the molecular mechanisms of the allosteric effects, we employed crystallography and biophysical methods. Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at 2.1 & Aring; resolution. Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of 1 mu M. The enhanced structural details now allow for unambiguous analysis of the MTPV-filled cavity intricately embedded within the enzyme. Finally, the structure suggests that MTPV binding induces an allosteric effect on the B-domain situated proximal to the active site. In summary, our study provides valuable insights into the allosteric regulation of PK by MTPV and paves the way for further structure-based drug optimization for therapeutic interventions in PK deficiency.