Isotope-tapping experiments with rabbit liver fructose bisphosphatase

Area of Science:

• Biochemistry
• Enzymology

Background:

• Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis.
• Understanding its catalytic mechanism is crucial for metabolic research.

Purpose of the Study:

• To investigate the role of metal ions and substrate binding in the catalytic mechanism of rabbit liver fructose 1,6-bisphosphatase.
• To elucidate the sequence of events during enzyme turnover.

Main Methods:

• Isotope-trapping experiments using mental-free rabbit liver fructose 1,6-bisphosphatase.
• Rapid-quench techniques to analyze enzyme-substrate interactions.

Main Results:

• D-fructose 1,6-bisphosphate dissociates from the enzyme before turnover.
• Structural Mn2+ ions are essential for productive substrate binding.
• An ordered ternary complex (enzyme-Mn2+-substrate) forms prior to catalysis.
• All four enzyme subunits are active during a single turnover.

Conclusions:

• Productive substrate binding to fructose 1,6-bisphosphatase necessitates the presence of structural Mn2+ ions.
• The catalytic mechanism involves an ordered binding of metal ion followed by substrate.
• The enzyme functions with all subunits active in a single turnover event.