Auramine O as a conformational probe to study glyceraldehyde 3-phosphate dehydrogenase.

Abstract

The interaction of yeast glyceraldehyde 3-phosphate dehydrogenase with a cationic fluorescent dye, auramine O, was studied by equilibrium dialysis, circular dichroism and fluorescence spectroscopy. Two equal and independent dye binding sites per tetrameric enzyme were detected with a dissociation constant of (4.5 ± 1) × 10−5 M. The maximum fluorescence of bound auramine O is at 515 nm, the excitation spectrum is characterized by the maxima at 380 nm and in the region of 460–470 nm. The complex of auramine O with the enzyme exhibits circular dichroism with a maximum at about 455 nm. The binding of NADH has no effect on the enzyme-dye interaction, whereas the oxidized coenzyme and nicotinamide mononucleotide displace auramine O from the protein. Nicotinamide mononucleotide was shown to induce conformational changes upon binding to the enzyme and to be capable of serving as a coenzyme in the dehydrogenase reaction. AMP, ADP and ADP-ribose form ternary complexes with the enzyme-dye binary complex. This results in a marked increase in fluorescence of the bound dye and was used to characterize the interaction between the enzyme and the adenine-containing NAD+ fragments. In the presence of glyceraldehyde 3-phosphate, the fluorescence of the bound auramine O is quenched, the dye-protein association constant remaining unchanged. Glyceraldehyde, a non-phosphorylated substrate analog, and glycerol 3-phosphate, a competitive inhibitor, are without any effect. Modification of the active-site cysteine-149 with iodoacetate does not affect the dye binding or fluorescence, whereas modification with iodoacetamide diminishes the fluorescence of the enzyme-bound dye without altering the dissociation constant. The effect of adenine-containing NAD+ fragments on the fluorescence of the enzyme-bound dye does not change upon modification of cysteine-149. Auramine O, added together with ADP-ribose, protects the apoenzyme against the inactivation due to modification of essential arginine residues. The effect is similar to that of NAD+ and is absent with auramine O alone or ADP-ribose alone.