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

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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
Volume370
Issue number2
DOIs
StatePublished - Jan 1 1989

Keywords

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

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