N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (K(i) = 0.24 mM) and a noncompetitive inhibitor with ATP (K(i) = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-biphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-biphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-biphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-biphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-biphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 μM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-biphosphatase are distinct and located in different regions of this bifunctional enzyme.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology