Characterization of recombinant fructose-1,6-bisphosphatase gene mutations: evidence of inhibition/activation of FBPase protein by gene mutation

Abstract

Specific residues of the highly regulated fructose-1,6-bisphosphatase (FBPase) enzyme serve as important contributors to the catalytic activity of the enzyme. Previous clinical studies exploring the genetic basis of hypoglycemia revealed two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia, linking the AMP-binding site to the active site of the enzyme. In the present study, a full kinetic analysis of similar mutants was performed. Kinetic results of mutants Y164A and M177A revealed an approximate two to three-fold decrease in inhibitory constants (Ki’s) for natural inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP) compared with the Wild-type enzyme (WT). A separate mutation (M248D) was performed in the active site of the enzyme to investigate whether the enzyme could be activated. This mutant displayed an approximate seven-fold increase in Ki for F2,6-BP. Interfacial mutants L56A and L73A exhibited an increase in Ki for F2,6-BP by approximately five-fold. Mutations in the AMP-binding site (K112A and Y113A) demonstrated an eight to nine-fold decrease in AMP inhibition. Additionally, mutant M248D displayed a four-fold decrease in its apparent Michelis constant (Km), and a six-fold increase in catalytic efficiency (CE). The importance—and medical relevance—of specific residues for FBPase structural/functional relationships in both the catalytic site and AMP-binding site is discussed.

In Silico Screening and Identification of Antidiabetic Inhibitors Sourced from Phytochemicals of Philippine Plants against Four Protein Targets of Diabetes (PTP1B, DPP-4, SGLT-2, and FBPase)

“…Twelve residues formed connections to all candidate ligands and the three references: Ala 24, Arg 140, Glu 29, Gly 21, Gly 26, Leu 30, Met 177, The 27, Thr 31, and Tyr 113. Of these 12, Met 177, Thr 31, and Tyr 113 are considered the most important residues due to their negative effect on the binding affinity when they are mutated [ 41 ]. Lys 112 is also among the key residues [ 41 ] and only missed interacting with C-0310.…”

“…Of these 12, Met 177, Thr 31, and Tyr 113 are considered the most important residues due to their negative effect on the binding affinity when they are mutated [ 41 ]. Lys 112 is also among the key residues [ 41 ] and only missed interacting with C-0310.…”

“…These residues encompass all four binding subsites in the protein’s catalytic domain, which could enhance the inhibitor’s potency and selectivity [ 33 , 42 ]. Lys 120 is a crucial PTP1B residue that significantly enhances ligand potency and selectivity, while Tyr 47 and Gln 262 are integral parts of the binding pocket for some compounds [ 30 , 41 ], so the residues’ strong, attractive interactions may also improve the inhibitor’s potency. Tyr 46, Arg 45, Ile 219, and Met 258 recorded the highest energy contribution in almost all top candidate ligands ( Figure S9 ).…”

“…Studies have pointed out the importance of Lys 112, Tyr 113, Met 177, and Thr 31 because of the observed decrease in inhibitory potency when these residues are mutated [ 40 , 41 ]. Met 177 displayed the most prominent contribution to binding, while Lys 112, Tyr 113, and Thr 31 had modest contributions ( Figure 14 ).…”

Fructose 1,6-bisphosphatase: getting the message across

“…Recent work by Topaz et al [45], published in Bioscience Reports extends our understanding of information transmission in mammalian FBPase. This study focused on seven variants: two which alter residues in a previously identified allosteric communication pathway and binding site for the allosteric inhibitor (4-(3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl)-thiazol-2-yl)-methanol (PFE; L56A and L73A); two affecting residues at the subunit interface (Y164A and M177A); two altering residues in the AMP binding site (K112A and Y113A) and one which changes a residue in a metal ion binding site (M248D).…”