Investigating the Origins of Cooperativity Utilizing an Archaeal Hyperthermophilic FBPase

Archeoglobus fulgidus, prokaryotic domain Archaea, possesses an unusual phosphatase, functioning as an inositol monophosphatase (IMPase), and fructose 1,6bisphosphatase (FBPase).  IMPase and FBPase cleave the phosphate group at C-1 at the carbon ring of inositol 1-phosphate to form di-myo-inositol, and fructose 6-bisphosphate respectively. Higher eukaryotes and various bacteria express both IMPases and FBPases as two separate gene products. The FBPase of the A. fulgidus show structural homology with mammalian FBPases in the active site, suggesting a possible evolutionary connection. In humans, IMPase is the drug target for manic-depressive illnesses and FBPase is the target for Type II diabetes. This study utilizes site directed mutagenesis, to investigate non-cooperative mechanisms (anti-cooperative) and to explore the origin of cooperative enzymes and how they may have evolved.  A. fulgidus FBPase was compared to pig kidney FBPase and human FBPase.   The results revealed a significant loss of affinity for FBP substrate without R167.  The structure of the R167A mutant was solved with molecular replacement using the wild-type enzyme. Comparison of wild type and mutant enzymes in our studies shows that R167 is necessary for tight binding of substrate.  Future studies to develop potent active site inhibitors, using the mammalian FBPase, will focus on substituting other functional groups for the phosphates at C1 and C6 of the FBP substrate fluorocarbons because the FBP enzyme can only cleave P-O bond but may bind tightly to these other electronegative functional groups, blocking substrate binding.   Potent inhibitors may be developed targeting the active site of this family of enzymes.