6-(p-toluidinyl)naphthalene-2-sulfonic acid as a fluorescent probe of yeast hexokinase: conformational states induced by sugar and nucleotide ligands.

Abstract

The fluorescent dye 6-(p-toluidinyl)naphthalene-2-sulfonic acid (2,6-TNS) has been shown to be a sensitive and nonperturbing probe of conformational states of yeast hexokinase. The binding of sugar ligands to hexokinase induced conformational states of the enzyme which could be distinguished by monitoring 2,6-TNS fluorescence and correlated well with their behavior during the catalytic reaction. The binding of five-carbon sugar inhibitors such as lyxose induced a conformational state of hexokinase that demonstrated a small quenching of 2,6-TNS fluorescence but an increased ability to bind metal-ligands when compared to free enzyme. The binding of good sugar substrates such as glucose produced a conformational state of hexokinase which demonstrated a large enhancement (37%) of bound 2,6-TNS fluorescence. This glucose-induced conformational state had an increased ability to bind metal-ATP ligands; however, the relative changes in the dissociation constants for the various metal-ATP ligands differ from those observed with hexokinase in the presence of lyxose. Hence, the lyxose-induced conformational state of hexokinase was concluded to be significantly different from the glucose-induced conformational state. The binding of poor sugar substrates such as 5-thioglucose induced a conformational state of hexokinase similar to the conformational state induced by glucose, but with a smaller enhancement of 2,6-TNS fluorescence (15%) and a lesser ability to increase the affinity for metal-ATP ligands. The six-carbon inhibitor with a bulky group on the 2-position, N-acetylglucosamine, gave minimal changes in 2,6-TNS fluorescence and effects on metal-nucleotide binding. These conformational states are interpreted in terms of the closure of the cleft between the two domains observed by X-ray crystallography. The binding of A1ATP to free hexokinase was not observed at concentrations up to 100 microM, which is consistent with the kinetic properties reported for this metal-ATP ligand. Although both CrATP and A1ATP have been reported to produce a slow burst-type transient in the progress curve of hexokinase, only CrATP demonstrated slow changes in 2,6-TNS fluorescence, indicating that the conformational state of hexokinase induced by A1ATP is different from the conformational state induced by CrATP.