Stewart H B, el-Maghrabi M R, Pilkis S J
J Biol Chem. 1985 Oct 25;260(24):12935-41.
We re-examined the kinetics of the bisphosphatase reaction of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase after depleting the enzyme of bound fructose 6-phosphate and found a hyperbolic dependence on fructose 2,6-bisphosphate at concentrations below 100 nM. The Michaelis constant was 4 nM, the Vmax was about 12 nmol X mg-1 X min-1 at 22 degrees C but the substrate inhibited at concentrations above 100 nM. Both phosphate and alpha-glycerol phosphate strongly inhibited phosphoenzyme formation and hydrolytic rate below 100 nM, but relieved the inhibition by substrate at higher concentrations probably by antagonizing substrate binding. A number of observations support the proposition that the phosphoenzyme is a necessary participant in catalysis. 1) The amount of phosphoenzyme measured during steady-state hydrolysis as a function of substrate concentration correlated with the velocity profile. 2) Rapid mixing experiments demonstrated that over a broad range of substrate concentrations phosphoenzyme formation was faster than the net rate of hydrolysis. 3) Both phosphate and alpha-glycerol phosphate inhibited the rate of phosphoenzyme formation and, at low substrate concentrations, reduced the steady-state phosphoenzyme levels. The latter correlated with inhibition of substrate hydrolysis. 4) Both phosphate and alpha-glycerol phosphate stimulate the rate of phosphoenzyme breakdown, consistent with their stimulation of substrate hydrolysis at high substrate concentrations. 5) The fractional rate of phosphoenzyme breakdown, which was pH and substrate dependent, multiplied by the amount of phosphoenzyme obtained in the steady state at that pH and substrate concentration approximated the observed rate of hydrolysis. We conclude that the phosphoenzyme is a reaction intermediate in the hepatic fructose-2,6-bisphosphatase reaction.
在去除大鼠肝脏6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶结合的6-磷酸果糖后，我们重新研究了该酶双磷酸酶反应的动力学，发现在果糖2,6-二磷酸浓度低于100 nM时，反应呈现双曲线依赖性。米氏常数为4 nM，在22℃时Vmax约为12 nmol·mg⁻¹·min⁻¹，但底物在浓度高于100 nM时会产生抑制作用。在100 nM以下，磷酸根和α-甘油磷酸根均强烈抑制磷酸酶的形成和水解速率，但在较高浓度时可能通过拮抗底物结合来缓解底物的抑制作用。多项观察结果支持磷酸酶是催化过程中必要参与者这一观点。1）在稳态水解过程中测量的磷酸酶量作为底物浓度的函数与速度曲线相关。2）快速混合实验表明，在广泛的底物浓度范围内，磷酸酶的形成比净水解速率更快。3）磷酸根和α-甘油磷酸根均抑制磷酸酶的形成速率，并且在低底物浓度下降低稳态磷酸酶水平。后者与底物水解的抑制相关。4）磷酸根和α-甘油磷酸根均刺激磷酸酶的分解速率，这与它们在高底物浓度下对底物水解的刺激作用一致。5）磷酸酶分解的分数速率依赖于pH和底物，乘以在该pH和底物浓度下稳态获得的磷酸酶量，近似于观察到的水解速率。我们得出结论，磷酸酶是肝脏果糖-2,6-二磷酸酶反应中的一个反应中间体。
