ANTAGONISTIC BINDING OF SUBSTRATES TO 8-PHOSPHOGLYCERATE KINASE MONITORED BY THE FLUORESCENT ANALOG 2'(3')-O-(2,4,6-TRINITROPHENYL)ADENOSINE 5'-TRIPHOSPHATE

The analogue of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), binds tightly to pig muscle 3-phosphoglycerate kinase. A dissociation constant K-d of 0.0095+/-0.0015 mM was determined by fluorimetric titration on the basis of 1:1 stoichiometry. TNP-ATP is a strong competitive inhibitor towards MgATP and MgADP with a K-i of 0.008+/-0.001 mM for both substrates. It is also a mixed-type inhibitor towards 3-phosphoglycerate with similar inhibition constants. Binding of TNP-ATP to 3-phosphoglycerate kinase is accompanied by a tenfold intensity increase and a blue shift of about 20 nm in its fluorescence emission spectrum and a shift of the pK of its trinitrophenyl group towards a more acidic pH. These findings suggest that the negatively charged trinitrophenyl group of TNP-ATP significantly contributes to the binding of the analogue. By stepwise replacement of the fluorescent TNP-ATP, the dissociation constants (K-d) for ADP and MgADP binding were determined and found to be 0.78+/-0.08 and 0.048+/-0.006 mM respectively, which are consistent with the values previously determined by equilibrium dialysis [Molnar and Vas (1993) Biochem J. 293, 595-599]. In similar competitive-titration experiments, ATP and MgATP did not completely substitute for TNP-ATP. For the fraction of the analogue that could be substituted, the dissociation constants for MgATP and ATP were estimated to be 0.27+/-0.09 and 0.33+/-0.15 mM respectively, close to the values determined by equilibrium dialysis. Using the same method, a significant weakening of binding of both (Mg)ADP and (Mg)ATP could be detected in the presence of 3-phosphoglycerate: their respective K-d values became 0.34+/-0.04 and 0.51+/-0.22 mM. The reciprocal effect, i.e. weakening of 3-phosphoglycerate binding in the presence of the nucleotide substrates, has been observed previously [Vas and Batke (1984) Eur. J. Biochem. 139, 115-123]. Similarly, a much weaker binding of (Mg)ATP could be observed in the presence of 1,3-bisphosphoglycerate (K-d = 2.30+/-0.68 mM). The possible reason for the mutual weakening of substrate binding is discussed in the light of the available structural data.