Nucleoside phosphorothioates as probes of the nucleotide binding site of brain pyridoxal kinase.

Abstract

N-Dansyl-2-oxopyrrolidine, a competitive inhibitor with respect to ATP, was used as a probe of the nucleotide binding site of pyridoxal kinase. It binds to an hydrophobic region of the catalytic site with a KD = 6 microM. Time emission anisotropy measurements yielded a rotational correlation time of 38 ns for the bound inhibitor. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the nucleotide binding site. Protein fluorescence quenching was used to determine the dissociation constants of the diastereomers of ATP beta S. Both diastereomers bind with a dissociation constant KD = 25 microM. The kinetic parameters Km and Vmax for the pyridoxal kinase reaction were determined for ATP and the diastereomers of ATP beta S in the presence of Mg(II), Co(II), Zn(II), and Cd(II). With Mg(II), pyridoxal kinase exhibits stereoselectivity for the A diastereomer of ATP beta S, Vmax ratio, A/B = 30. In the presence of Cd(II), the stereoselectivity is reversed and the B diastereomer of ATP beta S is the preferred substrate. As the divalent cations were varied in the series Mg(II), Co(II), Zn(II), and Cd(II), the A/B ratio was progressively lowered to the value of 0.2 found for Cd(II). These data indicate that the divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrates. The results obtained with the diastereomers of ATP alpha S suggest that pyridoxal kinase uses the divalent cation, delta,beta,gamma-bidentate nucleotide chelate as substrate.