Wheat germ phosphoglycerate mutase: purification, polymorphism, and inhibition

Area of Science:

• Biochemistry
• Enzymology

Background:

• Phosphoglycerate mutase (PGM) is crucial for glycolysis.
• Bisphosphoglycerate-independent PGM (iPGM) from wheat germ has unique properties.
• Efficient purification methods are essential for studying enzyme kinetics and structure.

Purpose of the Study:

• To develop an improved purification protocol for wheat germ iPGM.
• To characterize the purified enzyme's properties, including molecular forms and activity.
• To investigate enzyme inhibition mechanisms.

Main Methods:

• Enzyme purification using chromatography.
• Polyacrylamide gel electrophoresis (PAGE) under native and denaturing conditions.
• Isoelectric focusing (IEF).
• Enzyme activity assays.
• Inhibition studies with chelators and sulfhydryl reagents.

Main Results:

• A purification method yielding wheat germ iPGM with high specific activity (2,300 units/mg).
• Electrophoretic analysis revealed a single protein band (pI = 7.3) under native conditions.
• Denaturing PAGE identified two major enzyme forms (63 and 74 kDa) and minor high molecular mass components.
• Both major forms were catalytically active and capable of reversible monomer/dimer association.
• The enzyme exhibited time-dependent inhibition by polydentate chelators and sulfhydryl reagents.

Conclusions:

• The developed method provides highly purified wheat germ iPGM.
• Wheat germ iPGM exists as multiple molecular species, likely involved in monomer-dimer equilibria.
• The enzyme's active site is sensitive to sulfhydryl modification and metal chelation.