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首页> 外文期刊>Biochemistry >Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.
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Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.

机译：使用1H-15N异核多量子相干NMR光谱研究天冬氨酸转氨酶的活性位点。

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摘要

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.
机译：来自大肠杆菌的天冬氨酸转氨酶（一种88 kDa的酶）均匀且选择性地富含15N，并通过H2O中的异核多量子相干NMR光谱进行了研究。在酰胺，吲哚，咪唑和胍基团区域的NH质子的低场区域（1H方向的化学位移高于9.5 ppm）获得了良好的分辨率，并初步确定了几个共振。两个低场共振分别为12.6和11.36 ppm，似乎属于与氧或硫结合的质子。在11.78 ppm处，最弱磁场的共振发生在活性位点半胱氨酸192上，半胱氨酸192的肽质子与天冬氨酸199的带负电荷的羧基相距2.9A。当二羧酸抑制剂与酶的吡ido醛5'-磷酸（PLP）形式结合并且无机二价阴离子与吡ido胺5'-磷酸（PMP）形式结合时，观察到位点色氨酸140。我们根据可用的晶体学数据讨论这些惊人的差异。蛋白质的活性位点以及特定的抑制分子通常包含带负电荷的基团。这些可能能够形成与NH基团的氢键并将NH共振向低频移动到更不拥挤的区域，因此对于许多大蛋白而言更易于观察。我们的方法同时使用了HMQC光谱和NOE观测，应该得到广泛应用。

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