通过205Tl+核磁共振研究酵母丙酮酸激酶的构象变化。

Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.

Biochemistry. 1998 May 12;37(19):6967-74. doi: 10.1021/bi972454n.

The interaction of the monovalent cation with yeast pyruvate kinase (yPK) has been investigated by 205Tl+ NMR. TlNO3 activates yPK to 80-90% activity compared to KCl with an apparent Ka of 1.00 +/- 0.03 mM in the presence of 4 mM Mn(NO3)2 as the activating divalent cation. At higher concentrations of Tl+, enzyme inhibition is observed with an apparent KI of 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/T1 and 1/T2 values of 205Tl+ 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 for 205Tl+ longitudinal relaxation rates. The correlation time, tauc, for the Mn2+-205Tl+ interaction was estimated by a frequency dependence of 1/T1m for several enzyme complexes, and an average value of tauc was 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 A. Upon addition of phosphoenolpyruvate (PEP) to form the yPK-Tl-Mn-PEP complex, the inter-cation distance decreases to 6.1 +/- 0.3 A. 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 A. In the yPK-Tl-Mn-FBP complex, a Tl+-Mn2+ distance of 6.0 +/- 0.1 A is observed, indicating that FBP causes a conformational change at the active site in the absence of PEP. Analogous 205Tl 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.

通过205Tl+核磁共振研究了单价阳离子与酵母丙酮酸激酶（yPK）的相互作用。与氯化钾相比，硝酸铊可将yPK的活性激活至80 - 90%，在存在4 mM硝酸锰作为激活二价阳离子的情况下，其表观解离常数Ka为1.00±0.03 mM。在较高浓度的铊离子存在下，会观察到酶抑制现象，其表观抑制常数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）下测定了锰离子对存在yPK时205Tl+的1/T1和1/T2值的影响。弛豫速率的温度依赖性表明，205Tl+纵向弛豫速率处于快速交换条件。通过几种酶复合物的1/T1m频率依赖性估计了锰离子 - 205Tl+相互作用的相关时间tauc，其平均值确定为0.91 ns。根据锰离子对结合到yPK上的铊离子纵向（1/T1m）弛豫速率的顺磁贡献，计算了yPK活性位点处铊离子与锰离子之间的距离。对于脱辅基yPK复合物，铊离子到锰离子的距离为6.7±0.2 Å。加入磷酸烯醇丙酮酸（PEP）形成yPK - Tl - Mn - PEP复合物后，阳离子间距离减小至6.1±0.3 Å。加入变构激活剂果糖1,6 - 二磷酸（FBP）形成yPK - Tl+-Mn2+-PEP - FBP复合物时，金属间距离为6.2±0.2 Å。在yPK - Tl - Mn - FBP复合物中，观察到铊离子 - 锰离子距离为6.0±0.