Degree Type
Dissertation
Date of Award
2001
Degree Name
Doctor of Philosophy
Department
Biochemistry, Biophysics and Molecular Biology
First Advisor
Richard B. Honzatko
Abstract
Fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11, FBPase; Fructose 1,6-bisphosphate + H2O ⇔ Fructose 6-phosphate + Phosphate) is a key regulatory enzyme in gluconeogenesis. It is a homotetramer with subunit molecular mass of 37 kDa. There are two different quaternary conformations of FBPase: the R-state (the active conformation) and the T-state (the inactive conformation). FBPase is inhibited allosterically by AMP. FBPase requires divalent metal ions (Mg2+, Mn 2+, Zn2+, or Co2+) to hydrolyze fructose 1,6-bisphosphate. Some monovalent cations (K+, Rb+, Tl+ or NH3+) maximize the enzyme activity, whereas lithium ions inhibit the enzyme. Li+ inhibition of FBPase is similar to its effect on inositol monophosphatase, and, indeed FBPase and myo-inositol monophosphatase share a common polypeptide fold. FBPase is a target for the development of drugs in treating noninsulin dependent diabetes. A dynamic loop 52--72, revealed in a crystal structure of a product complex, and interacts with the active site of the enzyme. Loop 52--72 plays an important role in the allosteric mechanism, existing in two different conformations. A new divalent metal site and four monovalent metal sites were discovered. All of metal sites could be important in vivo. FBPase might select a combination of metals for activity in vivo. AMP competes with divalent metals and displaces loop 52--72 from the active site. Two distinct conformations for the 1-OH group of fructose 6-phosphate correspond to non-productive and productive states of ligation. Direct evidence for the existence of metaphosphate is presented in the active site of FBPase at near atomic resolution. The presence of the metaphosphate implies a dissociative mechanism in FBPase catalysis: The cleavage of the ester bond between atoms O-1 and P of the fructose 1,6-diphosphate precedes the nucleophilic attack of water.
DOI
https://doi.org/10.31274/rtd-180813-11525
Publisher
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Copyright Owner
Jun-yong Choe
Copyright Date
2001
Language
en
Proquest ID
AAI3016697
File Format
application/pdf
File Size
167 pages
Recommended Citation
Choe, Jun-yong, "X-ray crystallographic studies of the metal binding sites on recombinant porcine fructose-1,6-bisphosphatase " (2001). Retrospective Theses and Dissertations. 483.
https://lib.dr.iastate.edu/rtd/483
