Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme

Area of Science:

• Biochemistry
• Enzyme kinetics
• Protein conformational changes

Background:

• Glutamine synthetase (GS) from Escherichia coli is a crucial enzyme in nitrogen metabolism.
• Understanding the kinetic mechanisms of GS activation by divalent metal ions (Mg2+ and Mn2+) is vital for elucidating its catalytic function.
• Previous models often assumed simple bimolecular binding, but experimental data suggested a more complex process.

Purpose of the Study:

• To investigate the kinetic mechanisms of ADP and orthophosphate (Pi) binding to Mg2+- and Mn2+-activated unadenylylated glutamine synthetase.
• To determine the role of protein isomerization and substrate-induced conformational changes in the enzyme's catalytic cycle.
• To elucidate the reasons for the differential activity of Mn2+ versus Mg2+ in supporting the enzyme's biosynthetic function.

Main Methods:

• Kinetic analysis of protein fluorescence changes upon addition of ADP or Pi to enzyme-metal complexes.
• Determination of association constants (K1) for direct substrate binding.
• Calculation of thermodynamic parameters (ΔG°) and rate constants (k2, k-2) for substrate-induced conformational changes (isomerization).

Main Results:

• The kinetic patterns are inconsistent with simple bimolecular binding, supporting a mechanism involving direct substrate binding followed by conformational change (ES ⇌ ES*).
• Mn2+-activated enzyme exhibits slow ADP release from the Mn-enzyme-Pi-ADP complex, explaining its inability to support biosynthetic activity.
• Mg2+-activated enzyme shows faster ADP release, indicating this step is not rate-limiting for catalysis (kcat = 7.2 s-1).

Conclusions:

• Substrate binding to glutamine synthetase is followed by a conformational change, a key step in its catalytic mechanism.
• The slow dissociation of ADP from the Mn2+-enzyme complex is the primary reason for the reduced biosynthetic activity observed with Mn2+.
• The kinetic differences highlight the distinct roles of Mg2+ and Mn2+ in GS function, with Mg2+ facilitating more efficient product release.