Interaction of giyceraidehyde-3-phosphate dehydrogenase with amp as studied by means of a spin-labeled analog

Christine Karim, Reinhard Philipp, Wolfgang E. Trommer, Jane H. Park

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4 Scopus citations


The binding of a spin-labeled AMP analog to tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle is described. The spin label, perdeuterated and 15N-substituted 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, was attached to C-8 of AMP (C8-SL-AMP). Up to 8 equivalents of C8-SL-AMP bind per enzyme tetramer, i.e., 2 per monomer. Combining sites are the adenine subsite of the coenzyme-binding domain and the phosphate site. Glyceraldehyde 3-phosphate causes a conformational change in the enzyme that brings C8-SL-AMP molecules bound to adjacent R- axis-related subunits closer to one another by 0.2-0.3 nm and allows for spin-spin interaction between the nitroxide radicals. Similar, but less pronounced structural changes take place upon lowering the pH from 8 to 7. Addition of a single equivalent of NAD⊕ to a complex of the enzyme with 7.6 equivalents of C8-SL-AMP leads to the release of almost 4 C8-SL-AMP molecules. This supports our previous findings that binding of just one NAD⊕ molecule induces conformational changes in all four subunits.

Original languageEnglish (US)
Pages (from-to)1245-1252
Number of pages8
JournalBiological Chemistry Hoppe-Seyler
Issue number2
StatePublished - 1989

Bibliographical note

Funding Information:
The authors thank Mrs.Suzanne Brandon for her excellent technical assistance and Dr. Bruce H. Robinson for kindly providing us with his computer program for the simulation of ESR and saturation transfer ESR spectra in the slow motion regime. This work was supported by grants from Deutsche Forschungs-gemeinschaft andFondsderChemischenIndustrietoW.E.T.


  • 15N,2H-spin-label
  • Spin-labeled AMP
  • conformational changes
  • enzyme inhibition
  • glyceraldehyde-3-phosphate dehydrogenase
  • negative cooperativity


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