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.
Highlights
Fructose-1,6-bisphosphatase (FBPase) is a complex tetramer and a highly regulated enzyme
New mutant plasmids were designed for mutant protein M248D (Met248), L56A (Leu56), and L73 (Leu73), which were done via visual inspection of the active site and areas near tetramer interface (where PFE ({4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl}-methanol) binds; Figure 2A) in the graphics program Chimera to investigate if activity could be enhanced/altered as a result of mutation [17]
The pig kidney FBPase was used to perform these mutations, all of the mutated residues have shown to align with the human FBPase
Summary
Fructose-1,6-bisphosphatase (FBPase) is a complex tetramer and a highly regulated enzyme. FBPase activity is regulated synergistically by the allosteric inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP). FBPase functions in the degradation of fructose-1,6-bisphosphate (FBP), which hydrolyzes to fructose-6-phosphate (F6P) and inorganic phosphate [1]. FBPase is a key rate-controlling enzyme in the gluconeogenic pathway. Individuals with FBPase deficiency exhibit hypoglycemia and metabolic acidosis due to impaired gluconeogenesis. Hypoglycemia, an autosomal recessive disorder characterized by insufficient blood glucose levels, has been genetically linked to FBPase deficiency in clinical studies [2]. Since hypoglycemia has been found associated with FBPase mutations, impaired or mutated FBPase has proven to be a contributor to hypoglycemia [2]. It is plausible that a genetic link may exist between hyperglycemia and FBPase mutations
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call