Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Journal Article · Sun Nov 25 00:00:00 EST 1984 · J. Biol. Chem.; (United States)

OSTI ID:5273101

Sakakibara, R; Kitajima, S; Hartman, F C; Uyeda, K

The hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase were studied. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P and a noncompetitive inhibitor with ATP (K/sub i/ = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, 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/sup -1/. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate 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-P/sub 2/ in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activated fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P/sub 2/ saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K/sub 0.5/ of 0.8 ..mu..M. The reagent, however, has no effect on the fructose-6-P inhibition of phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme. 19 references, 9 figures, 1 table.

• Research Organization: Univ. of Texas Health Science Center, Dallas
• DOE Contract Number: AC05-84OR21400
• OSTI ID: 5273101
• Journal Information: J. Biol. Chem.; (United States), Vol. 259:22
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PHOSPHATASES
BIOCHEMICAL REACTION KINETICS
CONFIGURATION INTERACTION
PHOSPHATES
AMINES
ENZYME ACTIVITY
FRUCTOSE
PROTEOLYSIS
CARBOHYDRATES
CHEMICAL REACTIONS
DECOMPOSITION
ENZYMES
ESTERASES
HEXOSES
HYDROLASES
KETONES
KINETICS
MONOSACCHARIDES
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
REACTION KINETICS
SACCHARIDES
550200* - Biochemistry