Sakakibara R, Kitajima S, Hartman F C, Uyeda K
J Biol Chem. 1984 Nov 25;259(22):14023-8.
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 (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 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-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-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. 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-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.
为了研究双功能酶果糖-6-磷酸，2-激酶：果糖-2,6-二磷酸酶的磷酸己糖结合位点，对N-溴乙酰乙醇胺磷酸酯和3-溴-1,4-二羟基-2-丁酮1,4-二磷酸酯进行了测试。N-溴乙酰乙醇胺磷酸酯对果糖-6-磷酸是竞争性抑制剂（Ki = 0.24 mM），对ATP是非竞争性抑制剂（Ki = 0.8 mM）。该试剂使果糖-6-磷酸，2-激酶失活，但不使果糖-2,6-二磷酸酶失活，果糖-6-磷酸可阻止这种失活。失活反应遵循假一级动力学直至完成，随着N-溴乙酰乙醇胺磷酸酯浓度的增加，观察到速率饱和效应。产生半数最大失活的试剂浓度为2.2 mM，表观一级速率常数为0.0046 s-1。被N-溴乙酰乙醇胺-P烷基化的酶失去了超过90%的激酶活性，保留了几乎全部的果糖-2,6-二磷酸酶活性，并且其对果糖-6-磷酸的抑制作用未改变。3-溴-1,4-二羟基-2-丁酮1,4-二磷酸酯在正向反应中对果糖-6-磷酸，2-激酶也是果糖-6-磷酸的竞争性抑制剂，在反向反应中是果糖-2,6-二磷酸的竞争性抑制剂。该试剂抑制93%的果糖-6-磷酸，2-激酶，但使果糖-2,6-二磷酸酶激活3.7倍。3-溴-1,4-二羟基-2-丁酮1,4-二磷酸酯将果糖-2,6-二磷酸饱和动力学曲线从负协同性改变为正常的米氏动力学，K0.5为0.8 microM。然而，该试剂对磷酸酶的果糖-6-磷酸抑制作用没有影响。这些结果强烈表明，果糖-6-磷酸，2-激酶和果糖-2,6-二磷酸酶的磷酸己糖结合位点是不同的，且位于这种双功能酶的不同区域。
