Eisenstein E, Yu H D, Schwarz F P
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Baltimore.
J Biol Chem. 1994 Nov 25;269(47):29423-9.
Control of the regulatory enzyme threonine deaminase from Escherichia coli is achieved by isoleucine inhibition and valine activation. The mechanism by which these heterotropic effectors regulate the enzyme was investigated by measuring the binding of isoleucine and valine by spectroscopic, kinetic, calorimetric and equilibrium dialysis techniques. The addition of isoleucine or valine to threonine deaminase resulted in large changes in the intrinsic fluorescence of the two tryptophans per polypeptide chain. Slightly cooperative binding isotherms for isoleucine were obtained in potassium phosphate, pH 7.5, yielding an average dissociation constant of 4.91 microM, which was confirmed by equilibrium dialysis measurements. Valine binding was much more cooperative, and yielded an average dissociation constant of 122 microM. Titration calorimetry experiments indicated that cooperative heterotropic ligand binding was exothermic, and yielded a stoichiometry of four isoleucine bound per tetrameric enzyme, with an average enthalpy of -10.70 kcal/mol. Valine also bound to four sites per tetramer, with an average enthalpy of -7.45 kcal/mol. The effect of ligands on the fluorescence and circular dichroism spectra of the essential pyridoxal phosphate cofactor indicates that isoleucine and valine bind to effector sites that are distinct from the active sites in threonine deaminase. Shifts in the kinetic properties of threonine deaminase promoted by isoleucine and valine binding are to a first approximation consistent with analyses of effector binding isotherms in terms of a simple two-state model, and suggest that isoleucine regulates threonine deaminase by preferentially binding to the low activity T state, whereas valine binds preferentially to the high activity R state. Finally, analyses of heterotropic effector binding isotherms suggest that active site ligands may have significant affinity for the regulatory sites, which gives rise to underestimates for the allosteric equilibrium constants determined from substrate analog binding isotherms.
大肠杆菌中调节酶苏氨酸脱氨酶的调控是通过异亮氨酸抑制和缬氨酸激活来实现的。通过光谱、动力学、量热法和平衡透析技术测量异亮氨酸和缬氨酸的结合，研究了这些异向效应物调节该酶的机制。向苏氨酸脱氨酶中添加异亮氨酸或缬氨酸会导致每条多肽链上两个色氨酸的固有荧光发生很大变化。在pH 7.5的磷酸钾中获得了异亮氨酸的轻微协同结合等温线，平均解离常数为4.91 microM，这通过平衡透析测量得到了证实。缬氨酸的结合更具协同性，平均解离常数为122 microM。滴定热分析实验表明，协同异向配体结合是放热的，每个四聚体酶结合四个异亮氨酸的化学计量比为4，平均焓为-10.70 kcal/mol。缬氨酸也与每个四聚体的四个位点结合，平均焓为-7.45 kcal/mol。配体对必需的磷酸吡哆醛辅因子的荧光和圆二色光谱的影响表明，异亮氨酸和缬氨酸结合到与苏氨酸脱氨酶活性位点不同的效应物位点。异亮氨酸和缬氨酸结合促进的苏氨酸脱氨酶动力学性质的变化在一级近似下与基于简单二态模型的效应物结合等温线分析一致，表明异亮氨酸通过优先结合低活性T态来调节苏氨酸脱氨酶，而缬氨酸优先结合高活性R态。最后，对异向效应物结合等温线的分析表明，活性位点配体可能对调节位点具有显著亲和力，这导致从底物类似物结合等温线确定的变构平衡常数被低估。
