Conformational changes in yeast pyruvate kinase studied by 205Tl+ NMR.
Conformational changes in yeast pyruvate kinase studied by 205Tl+ NMR.
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通过 205Tl NMR 研究酵母丙酮酸激酶的构象变化。
DOI：
10.1021/bi972454n
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Nowak,T
中科院分区：
文献类型：
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作者：
Loria,JP;Nowak,T
用~（205）Tl ~+NMR研究了一价阳离子与酵母丙酮酸激酶（yPK）的相互作用。与KCl相比，TlNO 3激活yPK的活性为80 - 90%，在4 mM Mn（NO3）2作为激活二价阳离子的情况下，表观Ka为1.00 ± 0.03 mM。在较高浓度的Tl+下，观察到酶抑制，表观KI为180 ± 10 mM。抑制程度取决于二价阳离子的性质和浓度。在173.02 MHz（300 MHz，1H）和346.03 MHz（600 MHz，1H）下测定了Mn ~（2+）对~（205）Tl ~+的1/T_1和1/T_2值的影响。弛豫速率的温度依赖性表明，快速交换条件占主导地位为205 Tl+纵向弛豫速率。Mn 2 +− 205 Tl+相互作用的相关时间τc由几种酶复合物的1/T1 m频率依赖性估算，τ c的平均值为0.91 ns。根据Mn ~（2+）对与yPK结合的Tl ~+的纵向（1/T1 m）弛豫速率的顺磁贡献，计算了yPK活性位点处Tl ~+和Mn ~（2+）之间的距离。对于apo yPK复合物，Tl+至Mn 2+的距离为6.7 ± 0.2 μ m。当加入磷酸烯醇丙酮酸（PEP）形成yPK−Tl−Mn−PEP复合物时，阳离子间距离降低至6.1 ± 0.3 μ m。加入变构激活剂果糖1，6-二磷酸（FBP）形成yPK−Tl+− Mn 2 +−PEP−FBP复合物，金属间距离为6.2 ± 0.2 μ m。在yPK−Tl−Mn−FBP复合物中，观察到Tl+− Mn 2+距离为6.0 ± 0.1 μ m，表明在没有PEP的情况下，FBP会导致活性位点的构象变化。类似的205 T1 NMR实验与PEP（草酸盐，BrPEP）的竞争性抑制剂表明，这些配体不诱导相同的构象变化的生理底物和激活剂。还对非变构兔肌肉PK进行了类似的实验并进行了分析。
The interaction of the monovalent cation with yeast pyruvate kinase (yPK) has been investigated by205Tl+NMR. TlNO3activates yPK to 80−90% activity compared to KCl with an apparentKaof 1.00 ± 0.03 mM in the presence of 4 mM Mn(NO3)2as the activating divalent cation. At higher concentrations of Tl+, enzyme inhibition is observed with an apparentKIof 180 ± 10 mM. The extent of inhibition is dependent on the nature and concentration of the divalent cation. The effect of Mn2+on the 1/T1and 1/T2values of205Tl+in the presence of yPK was determined at 173.02 MHz (300 MHz,1H) and 346.03 MHz (600 MHz,1H). The temperature dependence of the relaxation rates indicates that fast exchange conditions prevail for205Tl+longitudinal relaxation rates. The correlation time, τc, for the Mn2+−205Tl+interaction was estimated by a frequency dependence of 1/T1mfor several enzyme complexes, and an average value of τcwas determined to be 0.91 ns. The distance between Tl+and Mn2+at the active site of yPK was calculated from the paramagnetic contribution of Mn2+to the longitudinal (1/T1m) relaxation rates of Tl+bound to yPK. For the apo yPK complex, the Tl+to Mn2+distance is 6.7 ± 0.2 Å. Upon addition of phosphoenolpyruvate (PEP) to form the yPK−Tl−Mn−PEP complex, the inter-cation distance decreases to 6.1 ± 0.3 Å. The addition of the allosteric activator fructose 1,6-bisphosphate (FBP) to form the yPK−Tl+−Mn2+−PEP−FBP complex gives an intermetal distance of 6.2 ± 0.2 Å. In the yPK−Tl−Mn−FBP complex, a Tl+−Mn2+distance of 6.0 ± 0.1 Å is observed, indicating that FBP causes a conformational change at the active site in the absence of PEP. Analogous205Tl NMR experiments with competitive inhibitors of PEP (oxalate, BrPEP) indicate that these ligands do not induce the same conformational changes as do the physiological substrates and activators. Similar experiments with the nonallosteric rabbit muscle PK were also performed and analyzed.