Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase.
Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase.
复制标题
天冬氨酸转氨甲酰酶上的配体结合位点之间的荧光能量转移。
DOI：
10.1021/bi00673a004
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发表时间：
1975
期刊：
影响因子：
2.9
通讯作者：
G. Hammes
中科院分区：
文献类型：
--
作者：
S. Matsumoto;G. Hammes
用荧光能量转移法测定了天冬氨酸转氨甲酰酶上几个位点之间的距离。使用荧光稳态和寿命技术。当催化亚基上的双氢吡喃是荧光供体基团时，与活性位点处的氨基共价结合的磷酸吡哆胺或与活性位点处的氨基非共价结合的8-苯胺基-1-萘磺酸盐是受体基团。色氨酸和活性位点之间的距离计算为2 e A，假设每个催化多肽链上只有一个色氨酸的荧光被受体猝灭，或27 A，假设催化链上的两个色氨酸被同等猝灭。磷酸吡哆胺标记物也用作荧光供体，与催化亚基的巯基结合的汞硝基苯酚作为能量受体。对于这对标记，活性位点被确定为非常接近于相同催化链上的巯基，并且26 A距离催化三聚体的其它链上的巯基。在以活性位磷酸吡哆胺为供体、活性位8-苯胺基-1-萘磺酸盐为受体的实验中，发现催化三聚体的活性位之间的距离为26 A，没有观察到活性位磷酸吡哆胺向调节位5 '-三磷酸胞苷的荧光胺衍生物的能量转移。这意味着这些基团在天然酶中被至少42 A分开。所有距离都是使用供体和受体偶极矩相对于供体荧光寿命快速旋转的假设来计算的。荧光偏振测量表明，这一假设不会产生显着的误差，在计算的距离。不同位点之间的距离与天冬氨酸转氨甲酰酶的亚基结构有关。
The method of fluorescence energy transfer is used to measure the distances between several sites on aspartate transcarbamylase. Both fluorescence steady-state and lifetime techniques are used. When the tryptophans on the catalytic subunit are the fluorescent donor groups, either pyridoxamine phosphate, covalently bound to an amino group at the active site, or 8-anilino-1-naphthalenesulfonate, noncovalently bound at the active site, is the acceptor group. The distance between tryptophan and the active site is calculated to be 2e A assuming that the fluorescence of only one tryptophan per catalytic polypeptide chain is quenched by the acceptor or 27 A assuming that both tryptophans on a catalytic chain are equally quenched. The pyridoxamine phosphate label is also used as the fluorescent donor with mercurinitrophenol bound to the sulfhydryl group of the catalytic subunit as the energy acceptor. For this pair of labels the active site is determined to be very close to the sulfhydryl group on the same catalytic chain and 26 A from the sulfhydryl groups on the other chains of the catalytic trimer. In experiments with pyridoxamine phosphate at the active site as the donor and 8-anilino-1-naphthalenesulfonate at the active site as the acceptor, a distance of 26 A between active sites of a catalytic trimer is found. No energy transfer is observed from pyridoxamine phosphate at the active site to a fluorescamine derivative of cytidine 5'-triphosphate at the regulatory site. This implies that these groups are separated by at least 42 A in the native enzyme. All of the distances are calculated using the assumption of rapid rotation of donor and acceptor dipole moments relative to the donor fluorescence lifetime. Fluorescence polarization measurements suggest this assumption does not produce a significant error in the calculated distances. The distances between the various sites are related to the subunit structure of aspartate transcarbamylase.