Conformational changes required for pyruvate kinase activity as modulated by monovalent cations

Area of Science:

• Biochemistry
• Enzymology

Background:

• Muscle pyruvate kinase (PK) is a key enzyme in glycolysis.
• Monovalent cations like K+ and NH4+ are known activators of PK.
• Understanding cation-binding mechanisms and conformational changes is crucial for enzyme function.

Purpose of the Study:

• To investigate the interaction of alkylamines with muscle pyruvate kinase.
• To elucidate the mechanisms of monovalent cation activation and conformational changes.
• To define conformational changes necessary for catalytic activity.

Main Methods:

• Kinetic studies of enzyme activity.
• Physical studies including ultraviolet difference spectroscopy.
• Water proton relaxation rate measurements to study substrate interaction.

Main Results:

• Monomethylammonium ion activates pyruvate kinase, while dimethyl- and trimethylammonium ions are competitive inhibitors.
• Ultraviolet difference spectroscopy revealed conformational changes induced by monomethylammonium and dimethylammonium ions.
• Alkylamines decreased the affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate by a factor of 5.
• The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex differed based on the monovalent cation present.

Conclusions:

• Conformational changes induced by certain alkylamines are necessary, but not sufficient, for active complex formation.
• The observed decrease in substrate affinity is not the sole reason for the lack of enzymic activity with certain alkylamines.
• The nature of the monovalent cation significantly influences the conformation of the ternary complex and enzyme activity.