Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts

Sci Rep. 2017 Jul 13;7(1):5343. doi: 10.1038/s41598-017-05557-w.


Enzymes sample multiple conformations during their catalytic cycles. Chemical shifts from Nuclear Magnetic Resonance (NMR) are hypersensitive to conformational changes and ensembles in solution. Phosphomannomutase/phosphoglucomutase (PMM/PGM) is a ubiquitous four-domain enzyme that catalyzes phosphoryl transfer across phosphohexose substrates. We compared states the enzyme visits during its catalytic cycle. Collective responses of Pseudomonas PMM/PGM to phosphosugar substrates and inhibitor were assessed using NMR-detected titrations. Affinities were estimated from binding isotherms obtained by principal component analysis (PCA). Relationships among phosphosugar-enzyme associations emerge from PCA comparisons of the titrations. COordiNated Chemical Shifts bEhavior (CONCISE) analysis provides novel discrimination of three ligand-bound states of PMM/PGM harboring a mutation that suppresses activity. Enzyme phosphorylation and phosphosugar binding appear to drive the open dephosphorylated enzyme to the free phosphorylated state, and on toward ligand-closed states. Domain 4 appears central to collective responses to substrate and inhibitor binding. Hydrogen exchange reveals that binding of a substrate analogue enhances folding stability of the domains to a uniform level, establishing a globally unified structure. CONCISE and PCA of NMR spectra have discovered novel states of a well-studied enzyme and appear ready to discriminate other enzyme and ligand binding states.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Enzyme Inhibitors / metabolism
  • Magnetic Resonance Spectroscopy*
  • Models, Molecular
  • Phosphoglucomutase / chemistry*
  • Phosphoglucomutase / metabolism*
  • Principal Component Analysis
  • Protein Binding
  • Protein Conformation
  • Pseudomonas / enzymology*
  • Sugar Phosphates / metabolism


  • Enzyme Inhibitors
  • Sugar Phosphates
  • Phosphoglucomutase