Evidence calcium pump binds magnesium before inorganic phosphate

Area of Science:

• Biochemistry
• Enzyme kinetics
• Membrane transport proteins

Background:

• P-type pumps are crucial for cellular ion transport.
• Understanding the binding order of substrates and cofactors is key to elucidating enzyme mechanisms.
• Previous studies suggested ordered binding in sodium pumps.

Purpose of the Study:

• To test the hypothesis that all P-type pumps, including the calcium pump, bind Mg2+ before Pi.
• To validate the use of a rate equation for ordered binding to interpret enzyme kinetics.
• To compare the kinetic properties of the calcium pump with the sodium pump.

Main Methods:

• Studied calcium pump-catalyzed (18)O exchange between inorganic phosphate (Pi) and water.
• Applied a rate equation for ordered binding of Mg2+ before Pi.
• Experimentally determined kinetic parameters such as dissociation constants and probabilities of phosphoenzyme formation.

Main Results:

• The ordered binding of Mg2+ before Pi fit the data, indicating Pi is the substrate and Mg2+ is an obligatory cofactor.
• The Mg2+ dissociation constant was similar to that of the sodium pump.
• The calcium pump exhibited a significantly higher Pi binding concentration and lower phosphoenzyme formation probability compared to the sodium pump, suggesting less efficient phosphoryl group transfer.

Conclusions:

• The calcium pump follows an ordered binding mechanism (Mg2+ then Pi), forming a ternary enzyme-metal-phosphate complex.
• This mechanism is consistent with the sodium pump and structural data from related enzymes.
• The calcium pump appears less efficient in catalyzing phosphoryl group transfer than the sodium pump.