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

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

S G Rhee, P B Chock

Biochemistry|April 20, 1976

View abstract on PubMed

Summary

The study reveals that glutamine synthetase kinetics involve substrate-induced conformational changes, not simple binding. Manganese (Mn2+) activation is hindered by slow ADP release, unlike magnesium (Mg2+), impacting enzyme function.

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.