Freye, C.; Miller, B. G.

Multi-functionality in extant enzymes, including the ability to transform multiple substrates, is thought to arise, in part, from conformational flexibility. The hexokinase protein family represents a classic model system for investigating the relationship between substrate specificity and conformational change. Within this family, human glucokinase (hGCK) displays notable degrees of conformational heterogeneity, including an intrinsically disordered loop. The extent to which these structural features contribute to the breadth of hGCK's substrate scope is unknown. Here, we investigate the substrate specificities of extant and ancestral glucokinases that span the evolutionary emergence of conformational heterogeneity in this family. We show that extant hGCK catalyzes the ATP-dependent phosphorylation of glucose, 2-deoxyglucose, mannose, glucosamine, fructose, allose and galactose with catalytic efficiencies ranging from 6.3 x 103 M-1sec-1 to 0.33 M-1sec-1. A glucokinase ancestor from early vertebrate evolution (vGCK), which also displays conformational heterogeneity and disorder, phosphorylates these same seven substrates with similar kcat/Km values. An antecedent, chordate glucokinase (cGCK), which displays reduced conformational heterogeneity and lacks intrinsic disorder, also transforms these same substrates, but with higher overall catalytic efficiencies and markedly lower Km values. Notably, however, the ratios of kcat/Km values for individual substrate pairs, which define specificity, are unchanged for all three enzymes. Our results demonstrate that substrate specificity is not correlated with conformational diversity in GCKs and support a model in which the differences in catalytic efficiencies of various substrates arise from differences in the ability to form the ground state enzyme-carbohydrate binary complex.