Friesen R H, Chin A J, Ledman D W, Lee J C
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1055, USA.
Biochemistry. 1998 Mar 3;37(9):2949-60. doi: 10.1021/bi971990c.
Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery. The muscle- and kidney-type isozymes are coded by the same gene. As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues. However, they exhibit very different regulatory behavior. In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia coli. The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure. Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg. This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors. The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator. In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor. These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and effectors communicate through this particular dimer-dimer interface. Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe.
丙酮酸激酶的组织特异性同工酶是阐明变构作用分子机制的特别有吸引力的系统。肌肉型和肾型同工酶由同一基因编码。由于选择性信使核糖核酸剪接，这两种一级序列仅相差少数几个残基。然而，它们表现出非常不同的调节行为。为了确定特定残基在赋予变构作用中的作用，编码兔肾型丙酮酸激酶的基因被克隆并在大肠杆菌中表达。重组兔肾型丙酮酸激酶（rRKPK）和重组兔肌肉型丙酮酸激酶（rRMPK）的一级结构在22个位置上不同，这些位置位于在RMPK结构中形成重要亚基间接触的区域。速度沉降和分析凝胶色谱研究表明，rRKPK经历可逆的二聚体⇌四聚体组装，平衡常数为28±3 mL/mg。这种亚基组装过程为阐明该二聚体界面在底物和变构效应剂结合时信号传递中的作用提供了机会。四聚体rRKPK的组装受到两种底物之一磷酸烯醇丙酮酸（PEP）或激活剂1,6-二磷酸果糖（FBP）结合的促进。相反，另一种底物二磷酸腺苷（ADP）或抑制剂L-苯丙氨酸（Phe）结合后，平衡向二聚体rRKPK移动。这些观察结果为丙酮酸激酶系统变构调节的分子机制提供了重要的新见解。首先，所有底物和效应剂都通过这个特定的二聚体-二聚体界面进行通信。其次，这些通信的热力学特征在两种底物之间以及激活剂FBP和抑制剂Phe之间在性质上是不同的。
