Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.
The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.
D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D005682:Gadolinium; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006801:Humans; D007461:Iodoacetates; D007811:Lanthanum; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding; D011817:Rabbits; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000032:analysis
D007461:Iodoacetates; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D007811:Lanthanum; D005682:Gadolinium; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine