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.