1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase.
1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase.
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与猪心 NADP 依赖性异柠檬酸脱氢酶结合的核苷酸的构象和环境的 1H 核磁共振研究。
DOI：
10.1021/bi00341a016
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Colman,RF
中科院分区：
文献类型：
--
作者：
Ehrlich,RS;Colman,RF
用质子核磁共振研究了辅酶NADP+和NADPH以及辅酶片段2'-磷酸腺苷5'-（二磷酸核糖）、腺苷2/、5/-二磷酸和2'-磷酸腺苷与猪心脏NADP+依赖的异柠檬酸脱氢酶的结合。烟酰胺v -核糖质子和2-烟酰胺酰胺质子之间的转移核过hauser增强（NOE）表明烟酰胺-核糖键呈反构象。对于所有的核苷酸，在腺嘌呤v -核糖质子和8-腺嘌呤环质子之间观察到核Overhauser效应，表明酶结合的核苷酸主要是合成酶- 9 -核糖键构象。在NADPH（而不是NADP+）中，在A2和N6的质子之间观察到转移的NOE，这意味着在NADPH的折叠酶结合形式中，腺嘌呤和nico -otinamide环之间的接近。与结合物种交换的自由核苷酸共振的线宽测量表明，解离速率从NADPH的< 7 s ' 1到腺苷2',5，-二磷酸的= 1600 s ' 1不等。底物异柠檬酸镁使NADPH的解离率提高了约10倍，但使二磷酸腺苷和2,5,5 -二磷酸腺苷的解离率降低了约10倍。这些效应与在类似条件下测得的平衡解离常数的变化是一致的。pH值为7.5时异柠檬酸脱氢酶的核磁共振谱在7.85 ~ 7.69之间有三个窄峰，它们随pH值的变化而移位，因此是由组氨酸的C-4质子引起的。其中pK=5.35的组氨酸受到NADP+和NADPH的干扰，但不受核苷酸片段的干扰，表明该组氨酸位于烟酰胺结合位点区域。对7.55的选择性辐照引起的核过hauser效应的观察表明，所有核苷酸的腺嘌呤环都接近非滴定组氨酸或芳香残基。此外，选择性照射酶谱的甲基区表明，腺嘌呤环靠近甲基侧链。底物异柠檬酸镁在这些蛋白质-核苷酸相互作用中没有可观察到的差异。在底物存在的情况下，酶核苷酸构象的改变导致亲和力的变化，必须涉及氨基酸侧链位置的微小变化或质子核磁共振光谱中不可见的基团的变化。
Department of Chemistry, University of Delaware, Newark, Delaware 19716 Received February 8, 1985 abstract: The binding of coenzymes, NADP+ and NADPH, and coenzyme fragments, 2'-phosphoadenosine 5'-(diphosphoribose), adenosine 2/, 5/-bisphosphate, and 2'-AMP, to pig heart NADP+-dependent isocitrate dehydrogenase has been studied by proton NMR. Transferred nuclear Overhauser enhancement (NOE) between the nicotinamide V-ribose proton and the 2-nicotinamidering proton indicates that the nicotinamide-ribose bond assumes an anti conformation. For all nucleotides, a nuclear Overhauser effect between the adenine V-ribose proton and 8-adenine ring proton is observed, suggesting a predominantly syn ade-nine-ribose bond conformation for the enzyme-bound nucleotides. Transferred NOE between the protons at A2 and N6 is observed for NADPH (but not NADP+), implying proximity between adenine and nic-otinamide rings in a folded enzyme-bound form of NADPH. Line-width measurements on the resonances of free nucleotides exchanging with bound species indicate dissociation rates ranging from< 7 s" 1 for NADPH to= 1600 s’1 for adenosine 2', 5,-bisphosphate. Substrate, magnesium isocitrate, increases the dissociation rate for NADPH about 10-fold but decreases the corresponding rate for phosphoadenosine diphosphoribose and adenosine 2,, 5,-bisphosphate about 10-fold. These effects are consistent with changes in equilibrium dissociation constants measured under similar conditions. The NMR spectrum of isocitrate dehydrogenase at pH 7.5 has three narrow peaks between 7.85 and 7.69 that shift with changes in pH and hence arise from C-4 protons of histidines. One of those, with pK=5.35, is perturbed by NADP+ and NADPH but not by nucleotide fragments, indicating that this histidine is in the region of the nicotinamide binding site. Observation of nuclear Overhauser effects arising from selective irradiation at 7.55 indicates proximity of either a nontitrating histidine or an aromatic residueto the adenine ring of all nucleotides. In addition, selective irradiation of the methyl region of the enzyme spectrum demonstrates that theadenine ring is close to methyl side chains. The substrate magnesium isocitrate produces no observable differences in these protein-nucleotide interactions. The alterations in enzyme-nucleotide conformation that result in changes in affinity in the presence of substrate must involve either small shifts in the positions of amino acid side chains or changes in groups not visible in the proton NMR spectrum.
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