Lin K, Li L, Correia J J, Pilkis S J
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
J Biol Chem. 1992 Sep 25;267(27):19163-71.
Rat liver fructose-2,6-bisphosphatase, which catalyzes its reaction via a phosphoenzyme intermediate, is evolutionarily related to the phosphoglycerate mutase enzyme family (Bazan, F., Fletterick, R., and Pilkis, S.J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646). Arg-7 and Arg-59 of the yeast phosphoglycerate mutase have been postulated to be substrate-binding residues based on the x-ray crystal structure. The corresponding residues in rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, Arg-257 and Arg-307, were mutated to alanine. The Arg257Ala and Arg307Ala mutants and the wild-type enzyme were expressed in Escherichia coli and then purified to homogeneity. Both mutant enzymes had identical far and near UV circular dichroism spectra and 6-phosphofructo-2-kinase activities when compared with the wild-type enzyme. However, the Arg257Ala and Arg307Ala mutants had altered steady state fructose-2,6-bisphosphatase kinetic properties; the Km values for fructose-2,6-bisphosphate of the Arg257Ala and Arg307Ala mutants were increased by 12,500- and 760-fold, whereas the Ki values for inorganic phosphate were increased 7.4- and 147-fold, respectively, as compared with the wild-type values. However, the Ki values for the other product, fructose-6-phosphate, were unchanged for the mutant enzymes. Although both mutants exhibited parallel changes in kinetic parameters that reflect substrate/product binding, they had opposing effects on their respective maximal velocities; the maximal velocity of Arg257Ala was 11-fold higher, whereas that for Arg307Ala was 700-fold lower, than that of the wild-type enzyme. Pre-steady state kinetic studies demonstrated that the rate of phosphoenzyme formation for Arg307Ala was at least 4000-fold lower than that of the wild-type enzyme, whereas the rate for Arg257Ala was similar to the wild-type enzyme. Furthermore, consistent with the Vmax changes, the rate constant for phosphoenzyme breakdown for Arg257Ala was increased 9-fold, whereas that for Arg307Ala was decreased by a factor of 500-fold, as compared with the wild-type value. The results indicate that both Arg-257 and Arg-307 interact with the reactive C-2 phospho group of fructose 2,6-bisphosphate and that Arg-307 stabilizes this phospho group in the transition state during phosphoenzyme breakdown, whereas Arg-257 stabilizes the phospho group of the ground state phosphoenzyme intermediate.
大鼠肝脏果糖-2,6-二磷酸酶通过磷酸化酶中间体催化反应，在进化上与磷酸甘油酸变位酶家族相关（巴赞，F.，弗莱泰里克，R.，和皮尔基斯，S.J.（1989年）《美国国家科学院院刊》86，9642 - 9646）。基于X射线晶体结构，酵母磷酸甘油酸变位酶的精氨酸-7和精氨酸-59被假定为底物结合残基。大鼠肝脏6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶中的相应残基，即精氨酸-257和精氨酸-307，被突变为丙氨酸。精氨酸257丙氨酸和精氨酸307丙氨酸突变体以及野生型酶在大肠杆菌中表达，然后纯化至均一。与野生型酶相比，两种突变酶具有相同的远紫外和近紫外圆二色光谱以及6-磷酸果糖-2-激酶活性。然而，精氨酸257丙氨酸和精氨酸307丙氨酸突变体改变了稳态果糖-2,6-二磷酸酶的动力学性质；与野生型值相比，精氨酸257丙氨酸和精氨酸307丙氨酸突变体的果糖-2,6-二磷酸的Km值分别增加了12500倍和760倍，而无机磷酸的Ki值分别增加了7.4倍和147倍。然而，突变酶的另一种产物果糖-6-磷酸的Ki值没有变化。尽管两种突变体在反映底物/产物结合的动力学参数上表现出平行变化，但它们对各自的最大速度有相反的影响；精氨酸257丙氨酸的最大速度比野生型酶高11倍，而精氨酸307丙氨酸的最大速度比野生型酶低700倍。预稳态动力学研究表明，精氨酸307丙氨酸的磷酸化酶形成速率比野生型酶至少低4000倍，而精氨酸257丙氨酸的速率与野生型酶相似。此外，与Vmax变化一致，与野生型值相比，精氨酸257丙氨酸的磷酸化酶分解速率常数增加了9倍，而精氨酸307丙氨酸的分解速率常数降低了500倍。结果表明，精氨酸-257和精氨酸-307都与果糖2,6-二磷酸的反应性C-2磷酸基团相互作用，并且精氨酸-307在磷酸化酶分解过程中在过渡态稳定该磷酸基团，而精氨酸-257稳定基态磷酸化酶中间体的磷酸基团。
