The Interaction of 8-Anilino-1-naphthalenesulfonate with Creatine Kinase EVIDENCE FOR COOPERATIVITY OF NUCLEOTIDE BINDING

Abstract Creatine kinase has one tight binding site (Kd = 310 µm) per subunit for 8-anilino-1-naphthalenesulfonate with an enhancement of fluorescence of ≃100 and a blue shift of the emission maximum of 30 nm. Nucleotide substrates non-competitively interact with the dye-binding site causing a 50% decrease in the fluorescence of the bound dye and a concomitant red shift of the emission maximum of 7 nm, while guanidino substrates alone do not affect the fluorescence of the bound dye. The fluorescence of the bound dye is used to determine the dissociation constants of nucleotide substrates from the enzyme in the presence or absence of other substrates or effectors, i.e. divalent metal ion or creatine. In the absence of a small planar anion, the presence of creatine or magnesium (or both) have no significant effect on the Kd of ADP from the enzyme, while in the presence of the nitrate anion creatine induces a drastic tightening and a negative cooperativity in the binding of ADP. With MgADP, the tightening of binding and the cooperativity are further accentuated. Modified inactive forms of the enzyme do not show the tightening nor the cooperativity of ADP or MgADP binding. These results are consistent with the hypothesis that the small planar anion (e.g. nitrate) is acting as an analog of a planar phosphoryl group, thus mimicking the transition state of the enzyme-substrate complex, and further suggest that two of the distinguishing features of this transition-state complex are a substantial interaction between the two protomeric subunits and the formation of a ligand from the protein to the divalent metal ion.