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

Abstract

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-bisphosphatasemore » are distinct and located in different regions of this bifunctional enzyme. 19 references, 9 figures, 1 table.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Univ. of Texas Health Science Center, Dallas
OSTI Identifier:
5273101
DOE Contract Number:  
AC05-84OR21400
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.; (United States)
Additional Journal Information:
Journal Volume: 259:22
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Sakakibara, R, Kitajima, S, Hartman, F C, and Uyeda, K. Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. United States: N. p., 1984. Web.
Sakakibara, R, Kitajima, S, Hartman, F C, & Uyeda, K. Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. United States.
Sakakibara, R, Kitajima, S, Hartman, F C, and Uyeda, K. Sun . "Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase". United States.
@article{osti_5273101,
title = {Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase},
author = {Sakakibara, R and Kitajima, S and Hartman, F C and Uyeda, K},
abstractNote = {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.},
doi = {},
url = {https://www.osti.gov/biblio/5273101}, journal = {J. Biol. Chem.; (United States)},
number = ,
volume = 259:22,
place = {United States},
year = {1984},
month = {11}
}