Mechanism of Substrate Recognition and Catalysis of the Haloalkanoic Acid Dehalogenase Family Member α-Phosphoglucomutase

Biochemistry. 2018 Jul 31;57(30):4504-4517. doi: 10.1021/acs.biochem.8b00396. Epub 2018 Jul 13.

Abstract

α-Phosphoglucomutase (αPGM), in its phosphorylated state, catalyzes the interconversion of α-d-glucose 1-phosphate and α-d-glucose 6-phosphate. The αPGM of Lactococcus lactis is a type C2B member of the haloalkanoic acid dehalogenase (HAD) enzyme family and is comprised of a Rossmann-fold catalytic domain and inserted α/β-fold cap domain. The active site is formed at the domain-domain interface. Herein, we report the results from a kinetic-based study of L. lactis αPGM catalysis, which demonstrate enzyme activation by autocatalyzed phosphorylation of Asp8 with αG1P, the intermediacy of αG1,6bisP in the phospho Ll-αPGM-catalyzed conversion of αG1P to G6P, and the reorientation of the αG1,6bisP intermediate via dissociation to solvent and rebinding. In order to provide insight into the structural determinants of L. lactis αPGM substrate recognition and catalysis, metal cofactor and substrate specificities were determined as were the contributions made by active-site residues toward catalytic efficiency. Lastly, the structure and catalytic mechanism of L. lactis αPGM are compared with those of HAD family phosphomutases L. lactis β-phosphoglucomutase and eukayotic α-phosphomannomutase to provide insight into the evolution of phosphohexomutases from HAD family phosphatases.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Catalytic Domain
  • Crystallography, X-Ray
  • Enzyme Activation
  • Glucose-6-Phosphate / metabolism
  • Glucosephosphates / metabolism
  • Kinetics
  • Lactococcus lactis / chemistry
  • Lactococcus lactis / enzymology*
  • Lactococcus lactis / metabolism
  • Models, Molecular
  • Phosphoglucomutase / chemistry
  • Phosphoglucomutase / metabolism*
  • Phosphorylation
  • Protein Conformation
  • Substrate Specificity

Substances

  • Glucosephosphates
  • Glucose-6-Phosphate
  • glucose-1-phosphate
  • Phosphoglucomutase