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

Abstract

GTP-l-fucose pyrophosphorylase(GFPP) catalyzes the reversible formation of the nucleotide-sugar GDP-beta-l-fucose from guanosine triphosphate and beta-l-fucose-1-phosphate. The enzyme functions primarily in the mammalian liver and kidney to salvage free fucose during the breakdown of glycoproteins and glycolipids. GFPP shares little primary sequence identity with other nucleotide-sugar metabolizing enzymes, and the three-dimensional structure of the protein is unknown. The enzyme does contain several sequences that could be nucleotide binding sites, but none of them are an exact match to consensus sequences. Using a combination of site-directed mutagenesis and UV photoaffinity cross-linking, we have identified five amino acid residues that are critical for catalysis. Some of these amino acids are found within the poorly conserved nucleotide binding consensus structures, while others represent new motifs. Two active site lysines can be cross-linked to photoaffinity probes. The site of cross-linking depends on the probe used. The identification of these critical residues highlights how distinct GFPP is from other nucleotide-sugar pyrophosphorylases.

“Reverse” Carbocyclic Fleximers: Synthesis of a New Class of Adenosine Deaminase Inhibitors

“…[11] The ability of Flex-GTP to interact with secondary amino acids within the binding site led to more favorable binding interactions, thereby increasing the affinity in GFPP when compared to GTP. [11,12] In addition to our findings, groups such as Hudson et al have pursued a series of analogues using click chemistry in which they have found interesting fluorescent properties. [13] It is important to note that, to date, no toxicity has been observed with any of the members of this interesting class of nucleosides.…”

Carbocyclic 5′-nor “reverse” fleximers. Design, synthesis, and preliminary biological activity

“…4,5 In addition to SAHase inhibition, it was also found that the triphosphate analogue of Flex-G was a significantly better substrate for GTP fucose pyrophosphorylase (GFPP) than the natural substrate GTP, and was able to retain full activity when key amino acid residues required for GTP recognition were mutated. 6,7 Notably, GTP was rendered completely inactive.…”

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.…”