Gidh-Jain M, Zhang Y, van Poelje P D, Liang J Y, Huang S, Kim J, Elliott J T, Erion M D, Pilkis S J, Raafat el-Maghrabi M
Department of Physiology and Biophysics, State University of New York at Stony Brook 11794-8861.
J Biol Chem. 1994 Nov 4;269(44):27732-8.
The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.
通过分子置换法，以猪肾酶与AMP的复合物为起始模型，利用X射线衍射确定了与AMP结合的人肝脏果糖-1,6-二磷酸酶的分子结构。在这两种序列中存在差异的34个氨基酸残基中，只有一个与AMP相互作用；猪肾中的Met30在人肝脏中为Leu30。通过该分析，利用聚合酶链反应对6个氨基酸残基侧链与AMP接触的位点（Ala24、Leu30、Thr31、Tyr113、Arg140和Met177）进行了突变。野生型和突变型在大肠杆菌中表达、纯化，并测定了它们的动力学性质。突变体的圆二色光谱与野生型酶的光谱无法区分。动力学分析表明，所有形式的酶都具有相似的周转数、对果糖2,6-二磷酸的Km值以及对果糖2,6-二磷酸的抑制常数。Leu30→Phe和Met177→Ala突变体对AMP抑制的表观Ki值与野生型酶相似，但Arg140→Ala和Ala24→Phe突变体的表观Ki值分别高出7至20倍。Thr31→Ser突变体对AMP的表观Ki值增加了5倍，而Thr31突变为Ala则使表观Ki值增加了120倍。即使在毫摩尔浓度的AMP下，也检测不到Tyr113→Phe突变体受到的AMP抑制。除了Thr31→Ala和Tyr113→Phe突变体的情况外，果糖2,6-二磷酸对突变体AMP抑制的增强程度与野生型酶相同。因此，Met177→Ala突变体表明，Cα以外的侧链对于AMP结合并非必需，而Leu30→Phe突变体保留了AMP与这些侧链的接触。Thr31、Tyr113和Arg140与AMP形成关键氢键，这与野生型酶中强烈的侧链相互作用一致。最后，在Thr31→Ala突变体中观察到的果糖2,6-二磷酸对AMP抑制没有任何影响，这可能是与这两种抑制剂协同作用机制相关的一个重要线索。
