Substrate Discrimination by the Human GTP Fucose Pyrophosphorylase

Abstract

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.

Identification of Catalytic Amino Acids in the Human GTP Fucose Pyrophosphorylase Active Site

“…All mutations were confirmed by DNA sequencing using the Applied Biosystems, Inc. model 373A automatic DNA sequencer. GFPP was purified as previously described (6).…”

“…The reaction is unusual in that, of the four canonical nucleoside triphosphates, only guanosine can be utilized efficiently to form a nucleotide-sugar (5). The GFPP active site though is more plastic than would have been imagined given the restricted base requirements (6). Surprisingly, the enzyme does not catalyze the formation of inorganic orthophosphate from the liberated pyrophosphate moiety as a way of energetically driving the reaction in the product formation direction.…”

1-Benzyl derivatives of 5-(arylamino)uracils as anti-HIV-1 and anti-EBV agents

“…[19][20][21][22][23][24] Similar to Tenofovir 25 and Etravirine, [26][27][28] both FDA-approved HIV-1-RT inhibitors, our compounds' inherent flexibility allows them to overcome resistance mechanisms related to point mutations. 29,30 The concepts of conformational flexibility and positional adaptability, or the ability to 'wiggle and jiggle' in a binding site when confronted with an active site mutation have now been shown [26][27][28] to be critical for avoiding resistance mechanisms for non-nucleoside HIV-1-RT inhibitors, thus provide additional impetus for our approach.…”

"Reverse Fleximers": Introduction of a series of 5-substituted carbocyclic uridine analogues

“…1 Conformational adaptability can allow for the engagement of secondary residues within the binding site that were not previously involved in the mechanism in order to retain its efficacy. 2,[4][5][6] The concept of nucleoside flexibility was previously introduced in our laboratories with the design of the "fleximers" [7][8][9][10][11] , where the purine base of traditional purine nucleosides has been split into its individual heterocyclic components. 10,11 Building on this principle, we have begun to explore new connectivities for these innovative nucleosides.…”