Schnackerz K D, Gracy R W
Institute of Physiological Chemistry, University of Würzburg, Federal Republic of Germany.
Eur J Biochem. 1991 Jul 1;199(1):231-8. doi: 10.1111/j.1432-1033.1991.tb16114.x.
We have explored the degree of independence of the two catalytic centers, interactions between the catalytic centers and the subunit-subunit contact sites, and different conformations of triosephosphate isomerase (TPI), by simultaneously employing irreversibly (covalent) and reversibly binding substrate analogues and monitoring their 31P-NMR resonances. 3-Chloroacetol phosphate (CAP) was bound to the active site by reaction with Glu165. The resulting, inactive (CAP-TPI)2 complex exhibited two distinct 31P-NMR resonances which were independent of pH and represent two conformational forms of the enzyme. Dissociation in guanidine hydrochloride followed by redimerization resulted in a single conformation. This was observed with the enzyme from chicken, rabbit and yeast. The inactive (CAP-TPI)2 dimer was mixed with native TPI, and dissociated/reassociated to form heterodimers (CAP-TPI)(TPI) in which one subunit contained the CAP label and the other subunit was unmodified. This hybrid migrated intermediate between the native and CAP-modified enzyme on nondenaturing PAGE. The heterodimer exhibited 50% the activity of the native dimer, but kinetic properties were otherwise indistinguishable. The reversibly binding transition state analogue, 2-phosphoglycolate (PGA), was used to probe the remaining vacant active site of the heterodimer. Bound PGA exhibited a pH-independent 31P-NMR resonance which was readily distinguishable from resonances of CAP-TPI and free PGA. No differences were observed in the binding of PGA to the vacant subunit of the heterodimer or the native dimer, further pointing to the independent nature of the two catalytic centers. However, the (CAP-TPI)(TPI) heterodimer was more susceptible to subunit dissociation in guanidine hydrochloride than the native dimer. Thus, it appears that the two active sites function completely independently of each other, but that the binding of CAP at the active center loosens the subunit-subunit contact. In addition, the two forms of the enzyme-inhibitor complex trapped by reaction with CAP may represent conformations with the hinged lid or flexible loop (residues 166-176) in the open and closed positions.
我们通过同时使用不可逆（共价）和可逆结合的底物类似物，并监测它们的31P-NMR共振，研究了两个催化中心的独立程度、催化中心与亚基-亚基接触位点之间的相互作用，以及磷酸丙糖异构酶（TPI）的不同构象。3-氯乙酰磷酸（CAP）通过与Glu165反应结合到活性位点。所得的无活性（CAP-TPI）2复合物表现出两个不同的31P-NMR共振，它们与pH无关，代表了酶的两种构象形式。在盐酸胍中解离后再二聚化产生单一构象。鸡、兔和酵母的酶都观察到了这种情况。将无活性的（CAP-TPI）2二聚体与天然TPI混合，解离/重新缔合形成异二聚体（CAP-TPI）（TPI），其中一个亚基含有CAP标记，另一个亚基未修饰。这种杂合体在非变性聚丙烯酰胺凝胶电泳上迁移于天然酶和CAP修饰酶之间。异二聚体的活性为天然二聚体的50%，但其动力学性质在其他方面无法区分。可逆结合的过渡态类似物2-磷酸乙醇酸（PGA）用于探测异二聚体中剩余的空活性位点。结合的PGA表现出与pH无关的31P-NMR共振，这与CAP-TPI和游离PGA的共振很容易区分。在PGA与异二聚体或天然二聚体的空亚基的结合中未观察到差异，这进一步表明了两个催化中心的独立性质。然而，（CAP-TPI）（TPI）异二聚体在盐酸胍中比亚天然二聚体更容易发生亚基解离。因此，似乎两个活性位点彼此完全独立发挥作用，但活性中心处CAP的结合会使亚基-亚基接触变松。此外，与CAP反应捕获的酶-抑制剂复合物的两种形式可能代表了铰链盖或柔性环（残基166-176）处于打开和关闭位置的构象。
