Catalytic cycling in beta-phosphoglucomutase: a kinetic and structural analysis.

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Lactococcus lactis beta-phosphoglucomutase (beta-PGM) is an enzyme in the haloalkanoic acid dehalogenase (HAD) superfamily.
• Beta-PGM catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P).
• Despite sharing a catalytic scaffold with phosphatases, beta-PGM functions as a mutase.

Purpose of the Study:

• To elucidate the structural basis for beta-PGM's functional diversification from a phosphatase to a mutase.
• To investigate the roles of Mg(2+) cofactor, Asp8 phosphorylation, and cap domain closure in beta-PGM catalysis.
• To understand substrate discrimination and the dominance of phosphomutase activity over phosphatase activity.

Main Methods:

• X-ray crystallography of Mg(2+)-beta-PGM complex at 1.90 A resolution.
• Analysis of previously reported related complex structures.
• Site-directed mutagenesis (D8A, D8E, E169A/D170A) and kinetic analyses.

Main Results:

• Asp8 is essential for nucleophilic catalysis, as D8A and D8E mutants lack activity.
• A single Mg(2+) coordination site accommodates water, phosphate, and the phosphorane intermediate during catalysis.
• Cap domain closure, potentially stabilized by Arg49-substrate interaction, is crucial for desolvation and catalysis.

Conclusions:

• Beta-PGM's mutase function is driven by structural adaptations within the HAD scaffold.
• Substrate-induced fit mechanism governs beta-PGM catalysis, favoring phosphomutase activity.
• Autophosphorylation of beta-PGM by beta-G1P explains the cellular origin of phospho-beta-PGM.