Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg(2+)

Area of Science:

• Biochemistry
• Enzymology
• Protein Science

Background:

• Free magnesium ions (Mg2+), beyond MgATP, influence numerous phosphorylase kinase (PhK) properties, including activity and molecular interactions.
• Mg2+ acts synergistically with Ca2+, another PhK activator, affecting enzyme characteristics.
• Previous research extensively documented Ca2+ effects on PhK, but comprehensive studies on Mg2+'s impact on its physical properties were lacking.

Purpose of the Study:

• To investigate the comprehensive effects of free Mg2+ on the physicochemical properties of phosphorylase kinase (PhK).
• To compare the Mg2+-induced changes in PhK properties under non-activating (pH 6.8) and activating (pH 8.2) conditions.
• To contrast the observed Mg2+ effects with previously reported Ca2+ effects on PhK.

Main Methods:

• Circular dichroism spectroscopy
• Zeta potential analyses
• Dynamic light scattering
• Second derivative UV absorption spectroscopy
• Negative stain electron microscopy
• Differential chemical crosslinking

Main Results:

• Mg2+ induced distinct changes in PhK properties at pH 6.8 and pH 8.2.
• The effects of Mg2+ on PhK showed both similarities and differences when compared to Ca2+.
• A key similarity was the significantly less negative zeta potential of PhK upon binding of both Mg2+ and Ca2+.

Conclusions:

• Free Mg2+ ions demonstrably alter the physical properties of PhK in a pH-dependent manner.
• The observed similarities in PhK response to Mg2+ and Ca2+ likely stem from their shared role as enzyme activators.
• The reduced negative zeta potential upon cation binding highlights a common mechanism for Mg2+ and Ca2+ activation of PhK.