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Characterization of Recombinant Fructose‐1,6‐Bisphosphatase (FBPase) Gene Mutations: Insights into Modulation of FBPase activity through Gene Mutation
Author(s) -
Topaz Gemma,
EpiterSmith Victor,
Emad Megan,
Ford Vanessa,
Daley Jadine,
Silva Christine,
Subramanian Manjula,
Sosa Harolin,
Stieglitz Kimberly
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.605.2
Subject(s) - fructose 1,6 bisphosphatase , enzyme , mutation , mutant , point mutation , wild type , microbiology and biotechnology , biochemistry , adenosine monophosphate , biology , gene , chemistry
Diabetes is a disease affecting how the body regulates glucose. Multiple factors contribute to Type II Diabetes resulting in hyperglycemia. Previous studies in patients with hypoglycemia identified two mutations in the coding region of the fructose‐1, 6‐bisphosphatase (FBPase) gene. Based on the mutations identified, the initial focus of our project was a full kinetic analysis of these mutations using the recombinant pig kidney FBPase enzyme. Results of this analysis showed an approximately 8‐fold decrease in the relative catalytic constant (K cat ) and catalytic efficiency (CE) compared to wild‐type enzyme (WTE). A separate mutation was done in the active site of the enzyme in order to investigate if the enzyme could be activated. Kinetic analysis of this mutation showed a 30‐fold increase in the relative K cat and catalytic efficiency (CE). Other mutations in the adenosine monophosphate (AMP) binding‐site (K112A and Y113A) showed marked decrease in AMP inhibition (AMP is the enzyme's natural inhibitor). Kinetic analysis of the enzyme in the absence of AMP resulted in maximum velocity (V max ) values for K112A and Y113A mutants that were similar to the wild‐type V max but which were elevated at higher concentrations of FBP substrate. This suggests that mutations of this type promote hyperglycemia by preventing AMP inhibition of the FBPase in vivo . Clinical implications for these results are discussed in relation to the project.