PHOSPHORUS NUCLEAR MAGNETIC-RESONANCE STUDIES OF PHOSPHOGLUCOMUTASE AND ITS METAL-ION COMPLEXES
PHOSPHORUS NUCLEAR MAGNETIC-RESONANCE STUDIES OF PHOSPHOGLUCOMUTASE AND ITS METAL-ION COMPLEXES
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DOI：
10.1016/0003-9861(77)90455-6
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发表时间：
1977-01-01
影响因子：
3.9
通讯作者：
GRUTZNER, JB
中科院分区：
文献类型：
--
作者：
RAY, WJ;MILDVAN, AS;GRUTZNER, JB
本文用傅里叶变换核磁共振波谱法研究了兔肌磷酸葡萄糖变位酶活性中心共价键合的磷酸基团的~（31）P核磁共振谱。在pD [氘离子浓度的倒数的对数]为7.9时，31P核的化学位移为3.8 ±。0.1 ppm低场从85%H3PO4;这一转变是接近磷酸丝氨酸（双阴离子形式）。质子去耦实验表明，酶磷酸基团的P与化学位移类似于磷酸丝氨酸的质子耦合。在D2O [氧化氘]，质子去耦，在1.6 mM磷酸酶的溶液中的纵向和横向的抗磁弛豫时间的比例产生的31 P核的酶的近似相关时间为10 - 7秒。这是在预期的整个蛋白质分子翻滚的值范围内，并表明共价连接的磷酸基团通过与相邻基团的非共价相互作用固定或冷冻在酶的活性位点。与此一致，酶磷酸的pKa显著低于磷酸丝氨酸。抗磁性激活剂Mg 2+的结合导致酶磷共振的化学位移从pD = 5.3到7.6的变化很小或没有变化，低场位移（-0.5 ±-0.5）。0.1 ppm），但高场位移（0.8 ± 0.1ppm）。0.1 ppm）的磷酸丝氨酸，这表明结合Mg 2+是不协调的酶磷酸。对直接协调的独立证据是由Ni 2+结合在活性位点上的酶磷的弛豫速率的顺磁效应。通过评估结合的Ni2+对所观察到的共振的纵向和横向弛豫速率的顺磁效应，并通过使用由Ni2+络合物诱导的H2O质子弛豫所确定的相关时间，Ni2+到磷的距离范围为4 - 6埃。计算了这些距离表明酶结合金属和酶磷酸基团之间的第二个球体相互作用。结合Ni~（2+）也明显降低~（31）P共振的积分强度。虽然这种强度下降的原因是不完全解释，目前的数据建立密切接近的结合金属离子和磷酸葡萄糖变位酶的活性位点磷酸丝氨酸。
The 31P NMR of the covalently bound phosphate group at the active site of rabbit muscle phosphoglucomutase was examined by means of Fourier transform NMR spectroscopy. At a pD [log of the reciprocal of the deuterium ion concentration] of 7.9, the chemical shift of the 31P nucleus is 3.8 .+-. 0.1 ppm downfield from 85% H3PO4; this shift is close to that of phosphoserine (dianionic form). Proton decoupling experiments suggest that the P of the enzymic phosphate group is coupled to protons with chemical shifts similar to those of phosphoserine. In D2O [deuterium oxide], with proton decoupling, the ratio of the longitudinal and transverse diamagnetic relaxaton times in solutions of 1.6 mM phosphoenzyme yields an approximate correlation time of 10-7 s for the 31P nucleus of the enzyme. This is within the range of values expected for tumbling of the entire protein molecule and suggests that the covalently attached phosphate group is immobilized or frozen at the active site of the enzyme by means of noncovalent interactions with adjacent groups. Consistent with this, the pKa of the enzymic phosphate is significantly lower than that of phosphoserine. Binding of the diamagnetic activator, Mg2+, causes little or no change in the chemical shift of the resonance of the enzymic phosphorus from pD = 5.3 to 7.6, a downfield shift (-0.5 .+-. 0.1 ppm) at pD = 8.6, but an upfield shift (0.8 .+-. 0.1 ppm) for that of phosphoserine, suggesting that bound Mg2+ is not coordinated to the enzymic phosphate. Independent evidence against direct coordination is provided by the paramagnetic effects of Ni2+ bound at the active site on the relaxation rates of the enzymic phosphorus. By assessing the paramagnetic effect of bound Ni2+ on both the longitudinal and transverse relaxation rates of the observed resonance, and by using correlation times determined for H2O proton relaxation induced by the Ni2+ complex, a range of Ni2+ to phosphorus distances of 4-6 .ANG. is calculated. These distances suggest a second sphere interaction between the enzyme-bound metal and the enzymic phosphate group. Bound Ni2+ also markedly decreases the integrated intensity of the 31P resonance. Although the reason for this intensity decrease is incompletely explained, the present data establish the close proximity of the bound metal ion and the active site phosphoserine on phosphoglucomutase.