Interaction of glyceraldehyde-3-phosphate dehydrogenase with AMP as studied by means of a spin-labeled analog.
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.
Karim
C
C
Fachbereich Chemie, Universität Kaiserslautern.
Philipp
R
R
Trommer
W E
WE
Park
J H
JH
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D003067:Coenzymes / Q000037:antagonists & inhibitors; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D005985:Glyceraldehyde / Q000031:analogs & derivatives; D005986:Glyceraldehyde 3-Phosphate / Q000037:antagonists & inhibitors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors; D007700:Kinetics; D008954:Models, Biological; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D013113:Spin Labels
D003067:Coenzymes; D003497:Cyclic N-Oxides; D013113:Spin Labels; D005986:Glyceraldehyde 3-Phosphate; C015527:4-amino-TEMPO; D005985:Glyceraldehyde; D000249:Adenosine Monophosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases