Residues of the ribose binding site are required for human ribokinase activity

Abstract

Ribokinase (RK) catalyzes the phosphorylation of D-ribose to ribose-5-phosphate, an essential metabolic intermediate for the pentose phosphate pathway, nucleotide biosynthesis, redox balance, and cellular energy metabolism. Despite its central physiological role, the structural and mechanistic bases of human RK activity remain incompletely defined. Here, we present the 2.1 Å resolution crystal structure of human RK bound to ADP and Mg²⁺, revealing a conserved dimeric architecture characteristic of the PfkB family. A conserved β-clasp motif at the dimer interface stabilizes asymmetric conformations between protomers, supporting dynamic active-site gating. Mutagenesis of key ribose-coordinating residues—Asp27, Lys54, Asn57, Glu154, and Ala181—completely abolished catalytic activity, underscoring the essential roles of these residues in substrate binding and positioning. Kinetic analysis showed that the partially active E154A mutant had impaired substrate affinity and catalytic efficiency, indicating that E154 contributes to ribose coordination and active-site geometry. Molecular dynamics simulations further demonstrated that mutations of ribose-coordinating residues disrupt ribose retention and accelerate active-site opening, thereby destabilizing the catalytic pocket. Complementary thermodynamic and kinetic stability measurements revealed that the introduction of E154A reduced both the melting temperature and enzymatic half-life of RK under heat stress, linking structural flexibility to functional robustness. Collectively, these observations define a finely tuned network of ribose-binding interactions that are essential for catalytic activity, conformational regulation, and thermal stability. This work establishes a mechanistic framework for human RK function to support its exploration as a therapeutic target in metabolic and cardiovascular disorders with perturbed ribose metabolism.