GTP-l-fucose pyrophosphorylase (GFPP, E. C. 2.7.7.30) catalyzes the reversible condensation of guanosine triphosphate and beta-l-fucose-1-phosphate to form the nucleotide-sugar GDP-beta-l-fucose. The enzyme functions primarily in the mammalian liver and kidney to salvage free l-fucose during the breakdown of glycolipids and glycoproteins. The mechanism by which this protein discriminates between substrate and nonsubstrate molecules has been elucidated for the first time in this study. The ability of GFPP to form nucleotide-sugars from a series of base-, ribose-, phosphate-, and hexose-modified precursor molecules has revealed that the enzyme active site senses a series of substrate substituents that drive substrate/nonsubstrate discrimination. These substituents alter the ability of the precursor molecule to interact with the enzyme, as measured by either changes in the Michaelis constant, K(m), the binding affinity, K(a), or through changes in enzymatic turnover, k(cat). In this work, the combined substrate binding and enzyme analysis has revealed that the nature of the purine base is the major determinant in substrate specificity, followed by the nature of the hexose-1-P, and finally by the ribose moiety. Binding is enthalpy-driven and does not involve proton transfer. For the majority of nucleotide-sugar analogues, binding to GFPP is entropically unfavorable; however, surprisingly, a few of the substrate analogues tested bind to GFPP with a favorable entropic term.