Conformational Expansion Underlies the Evolutionary Emergence of Redox Sensitivity in Vertebrate Glucokinases

Glucokinase (GCK) catalyzes the first step of glycolysis in pancreatic β-cells, where it functions as the body's primary glucose sensor. GCK is extremely sensitive to oxidative inactivation, both in vivo and in vitro . This characteristic provides a mechanism to regulate GCK activity via alterations in the cellular redox environment. To understand the molecular and evolutionary origins of redox regulation, we characterized the sensitivity to oxidative inactivation of four extant GCKs and five ancestral GCKs produced from a recent phylogenetic analysis of the vertebrate family. We find that two invertebrate GCKs are significantly less sensitive to oxidative inactivation compared to their vertebrate counterparts. We also demonstrate that an ancestral GCK from chordates (cGCK) is insensitive to oxidative inactivation, whereas an ancestral GCK from early vertebrates (vGCK) displays a degree of redox responsiveness comparable to the extant human enzyme. The redox insensitive cGCK ancestor lacks cysteine residues at two positions, Cys230 and Cys461, that are conserved in all redox sensitive ancestral and extant enzymes. We find that installation of cysteines at these positions is insufficient to install redox sensitivity into cGCK. Our data demonstrate that the appearance of redox responsiveness in GCKs coincides with an expansion in the conformational landscape of the protein that occurred during early vertebrate evolution. These observations support a model in which the emergence of redox sensitivity required the ability to sample a unique super-open conformation, an event that also facilitated the emergence of two orthogonal GCK regulatory strategies, allosteric regulation by substrate glucose and an inhibitory interaction with the glucokinase regulatory protein.