Magnetic resonance studies of manganese (II) binding sites of pyruvate kinase. Temperature effects and frequency dependence of proton relaxation rates of water.
Magnetic resonance studies of manganese (II) binding sites of pyruvate kinase. Temperature effects and frequency dependence of proton relaxation rates of water.
复制标题
丙酮酸激酶的锰 (II) 结合位点的磁共振研究。
DOI：
--
复制
发表时间：
1970
影响因子：
4.8
通讯作者：
M. Cohn
中科院分区：
文献类型：
--
作者：
J. Reuben;M. Cohn
用电子顺磁共振监测溶液中游离锰(II)的量，研究了兔肌丙酮酸激酶-锰(II)二元络合物在5-37℃温度范围内的化学计量比和离解常数。结果被解释为酶的两种构象的平衡混合物，一种有四个结合锰(II)的位置(“活性”)，另一种不结合锰(II)的(“非活性”)。这种平衡受到单价离子的影响；与酶惰性的四甲基铵离子相比，丙酮酸激酶反应的激活剂钾稳定了“活性”形式。本文用脉冲核磁共振技术测量了水在丙酮酸激酶和锰(II)溶液中的纵向质子弛豫速率随温度(5-37°)和频率(8-60.0 MHz)的变化，以深入了解弛豫机理和确定蛋白质与金属的配位数。根据所罗门-Bloembergen-Morgan方案，分析了结合的Mn(II)离子相对于Mn(II)水离子高度增强的质子弛豫速率。详细介绍了分析的基本原理和方法。与Mn(II)水离子与水质子相互作用的相关关联时间是旋转时间从而与频率无关不同，我们发现在结合的Mn(II)附近的水的质子驰豫的关联时间至少部分地依赖于频率。频率相关成分与结合的Mn(II)的纵向电子自旋弛豫时间有关，另一成分与水分子在第一水化球体中的平均停留时间有关，即水配体交换率的倒数。结果表明，丙酮酸激酶与Mn(II)配位的水分子数为3个，推测该酶可能为Mn(II)提供了3个配位体，从而解释了以前用蛋白质差示光谱检测到的二价金属离子结合引起的构象变化。虽然用四甲基铵取代钾离子时，二元锰(II)-酶络合物的结合常数不同，但对水的质子弛豫速率的提高与一价离子是不变的。
Abstract The stoichiometry and dissociation constant of the binary rabbit muscle pyruvate kinase-manganese(II) complex have been investigated in the temperature range 5–37° by monitoring the amount of Mn(II) free in solution utilizing electron paramagnetic resonance spectrometry. The results have been interpreted in terms of an equilibrium mixture of two conformational forms of the enzyme, one which has four binding sites for Mn(II) ("active") and another which does not bind Mn(II) ("inactive"). The equilibrium is affected by monovalent ions; potassium, an activator of the pyruvate kinase reaction, stabilizes the "active" form as compared to the enzymatically inert tetramethylammonium ion. Longitudinal proton relaxation rates of water in solutions of pyruvate kinase and Mn(II) have been measured by pulsed nuclear magnetic resonance spectrometry as a function of temperature (5–37°) and frequency (8 to 60.0 MHz) in order to gain some insight into the mechanism of relaxation and to determine the number of protein ligands to the metal. The proton relaxation rates due to bound Mn(II), highly enhanced relative to the Mn(II) aquo-ion, have been analyzed according to the Solomon-Bloembergen-Morgan scheme. The rationale and method of analysis are presented in detail. Unlike the Mn(II) aquo-ion for which the relevant correlation time for the interaction between Mn(II) and the water protons is the rotational time and consequently frequency-independent, it has been found that the relevant correlation time for proton relaxation of water in the vicinity of bound Mn(II) is, at least in part, frequency-dependent. The frequency-dependent component is identified with the longitudinal electron spin relaxation time of the bound Mn(II) and the other component with the mean residence time of a water molecule in the first hydration sphere, i.e. the reciprocal of the water ligand exchange rate. From the results the number of water molecules coordinated to Mn(II) bound to pyruvate kinase has been estimated to be 3. It may be concluded that the enzyme probably provides three ligands for Mn(II) and thus the conformational changes previously detected by protein difference spectroscopy due to the binding of a divalent metal ion may be rationalized. Although the binding constant of the binary Mn(II)-enzyme complex varies upon substitution of tetramethylammonium for potassium ion, the enhancement of the proton relaxation rate of water for the binary complex is invariant with monovalent ion.