Substrate channeling in glycolysis: a phantom phenomenon

Area of Science:

• Biochemistry
• Enzymology
• Metabolic pathways

Background:

• Glycolytic enzymes are proposed to form multienzyme complexes for efficient metabolite transfer.
• Direct transfer of NADH between glycerol-3-phosphate dehydrogenase (GPDH) and L-lactate dehydrogenase (LDH) has been suggested.
• Previous evidence supporting direct transfer has been questioned due to kinetic misinterpretations.

Purpose of the Study:

• To investigate the mechanism of NADH transfer between GPDH and LDH.
• To determine if GPDH and LDH form stable multienzyme complexes.
• To re-evaluate the kinetics of NADH binding and displacement between these enzymes.

Main Methods:

• Detailed kinetic studies of NADH binding to GPDH and LDH.
• Experiments measuring the displacement of enzyme-bound NADH by GPDH or LDH.
• Sedimentation equilibrium, gel filtration, and tracer experiments to detect enzyme complex formation.

Main Results:

• Kinetic patterns of NADH displacement are consistent with a dissociative mechanism, not direct transfer.
• Theoretical analysis suggests direct transfer is only possible if extensive enzyme complexation occurs (approx. 90%).
• Experimental data show no evidence of GPDH and LDH complex formation at relevant enzyme concentrations.

Conclusions:

• Glycerol-3-phosphate dehydrogenase (GPDH) and L-lactate dehydrogenase (LDH) do not form multienzyme complexes.
• The transfer of NADH between GPDH and LDH proceeds via a dissociative mechanism.
• The findings refute the hypothesis of direct metabolite transfer through stable enzyme complexes in this system.