Time-resolved NMR: extracting the topology of complex enzyme networks

Biophys J. 2010 Oct 6;99(7):2318-26. doi: 10.1016/j.bpj.2010.08.014.

Abstract

The use of nondestructive NMR spectroscopy for enzymatic studies offers unique opportunities to identify nearly all enzymatic byproducts and detect unstable short-lived products or intermediates at the molecular level; however, numerous challenges must be overcome before it can become a widely used tool. The biosynthesis of acetyl-coenzyme A (acetyl-CoA) by acetyl-CoA synthetase is used here as a case study for the development of an analytical NMR-based time-course assay platform. We describe an algorithm to deconvolve superimposed spectra into spectra for individual molecules, and further develop a model to simulate the acetyl-CoA synthetase enzyme reaction network using the data derived from time-course NMR. Simulation shows indirectly that synthesis of acetyl-CoA is mediated via an enzyme-bound intermediate (possibly acetyl-AMP) and is accompanied by a nonproductive loss from an intermediate. The ability to predict enzyme function based on partial knowledge of the enzymatic pathway topology is also discussed.

Publication types

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

MeSH terms

  • Acetate-CoA Ligase / chemistry*
  • Acetate-CoA Ligase / metabolism
  • Algorithms
  • Arabidopsis / enzymology*
  • Biocatalysis
  • Least-Squares Analysis
  • Magnetic Resonance Spectroscopy
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism
  • Time Factors

Substances

  • Multienzyme Complexes
  • Acetate-CoA Ligase