Antagonistic binding of substrates to 3-phosphoglycerate kinase monitored by the fluorescent analogue 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate

Abstract

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 Kd 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 Ki 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 (Kd) 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 [Molnár 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 Kd 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 (Kd = 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.