Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase.
Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase.
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磷酸葡萄糖变位酶金属离子结合位点的多核磁共振研究。
DOI：
10.1021/bi00331a021
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Markley,JL
中科院分区：
文献类型：
--
作者：
Rhyu,GI;RayJr,WJ;Markley,JL
化学和生物科学系，普渡大学，西拉斐特，印第安纳州47907接收1984年7月30日摘要：金属结合在激活位点的兔肌肉磷酸葡萄糖变位酶已被研究的31 P，7 Li，和113 Cd NMR光谱。一个7 Li NMR信号的binaryLi+复合物的磷酸酶没有观察到可能是因为快速横向松弛的结合离子由于与游离Li+的化学交换。磷酸酶-Li ~+-葡萄糖6-磷酸三元络合物在动力学上更稳定，并在LiCl的-0.24ppm处产生一个与结合Li ~+分离良好的峰，其线宽为5 Hz，在78 MHz处的弛豫时间为0.51± 0.07s。相反，当葡萄糖1-磷酸结合时，结合的7 Li+的化学位移为-0.13 ppm;在脱磷酸酶和葡萄糖二磷酸的Li+络合物中，在-0.08 ppm处出现部分加宽的7 Li+峰。因此，结合的金属离子在这三种三元络合物中的每一种中都具有稍微不同的环境。磷酸酶的二元Cd 2+络合物的U3 Cd NMR信号出现在相对于Cd（C104）2的22 ppm处，在44.4 MHz处具有20 Hz的线宽。底物的结合和脱磷酸酶和葡萄糖二磷酸盐的Cd 2+复合物的形成将113 Cd NMR信号拓宽至70 Hz并将其移位至75 ppm。沿着加入底物后的53 ppm的低场位移与 * H NMR数据表明，在酶促反应的一些中间点，二元络合物中的Cd 2+的一个氧配体被氮配体取代。除了Li+和Cd 2+在金属离子活化位点的结合之外，在结合底物的存在下，观察到任一金属离子在辅助位点的结合较弱。结合的Li+或Cd 2+在弱位点的Cd 2 +-dephosphoenzyme-bisphosphate复合物的结果在类似的化学位移变化的31 P NMR峰的bisphosphate，大概是凭借在酶的构象变化。在没有结合底物的情况下，没有观察到辅助结合对酶磷酸盐的影响。从Li+或Cd 2+结合在弱站点的信号没有观察到在7 Li NMR或I13 Cd NMR的研究，显然是因为快速的横向弛豫产生的化学交换过程。虽然单价Li+在二价金属离子激活位点的结合可能是磷酸葡萄糖变位酶所特有的，但目前的研究表明，在其他酶系统中使用Li+作为二价金属离子结合位点的探针可能是富有成效的。
Departments of Chemistry and Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received July 30, 1984 abstract: Metal binding at the activating site of rabbit muscle phosphoglucomutase has been studied by 31P, 7Li, and 113Cd NMR spectroscopy. A 7Li NMR signal of the binaryLi+ complex of the phosphoenzyme was not observed probably because of rapid transverse relaxation of the bound ion due to chemical exchange with free Li+. The phosphoenzyme-Li+-glucose 6-phosphate ternary complex is more stable, kinetically, and yields a well-resolved peak from bound Li+ at-0.24 ppm from LiCl with a line width of 5 Hz and a, relaxation time of 0.51±0.07 s at 78 MHz. When glucose 1-phosphate was bound, instead, the chemical shift of bound 7Li+ was-0.13 ppm; and in the Li+ complex of the dephosphoenzyme and glucose bisphosphate a partially broadened 7Li+ peak appeared at-0.08 ppm. Thus, the bound metal ion has a somewhat different environment in each of these three ternary complexes. The U3Cd NMR signal of the binary Cd2+ complex of the phosphoenzyme appears at 22 ppm relative to Cd (C104) 2 with a line width of 20 Hz at 44.4 MHz. Binding of substrate and formation of the Cd2+ complex of the dephosphoenzyme and glucose bisphosphate broaden the 113Cd NMR signal to 70 Hz and shift it to 75 ppm. The 53 ppm downfield shift upon the addition of substrate along with* H NMR data suggests that one oxygen ligand to Cd2+ in the binary complex is replaced by a nitrogen ligand at some intermediate point in the enzymic reaction. In addition to the binding of Li+ and Cd2+ at the activating site for metal ions, weaker binding of either metal ion at an ancillary site was observed in the presence of bound substrate. Binding of either Li+ or Cd2+ at the weak site of the Cd2+-dephosphoenzyme-bisphosphate complex results in similar chemical shift changes in the 31P NMR peaks of the bisphosphate, presumably by virtue of a conformational change in the enzyme. No effect of ancillary binding was observed on the enzymic phosphate in the absence of bound substrate. Signals from Li+ or Cd2+ bound at the weak site were not observed in the 7Li NMR or I13Cd NMR studies, apparently because of rapid transverse relaxation resulting from a chemical exchange process. Although the binding of the monovalent Li+ at the activating site for divalent metal ions may be peculiar to phosphoglucomutase, the present studies suggest that the use of Li+ as a probe of the binding site for bivalent metal ions inother enzymic systems might prove fruitful.