Binding of the oxidized, reduced, and radical flavin species to chorismate synthase. An investigation by spectrophotometry, fluorimetry, and electron paramagnetic resonance and electron nuclear double resonance spectroscopy.
Binding of the oxidized, reduced, and radical flavin species to chorismate synthase. An investigation by spectrophotometry, fluorimetry, and electron paramagnetic resonance and electron nuclear double resonance spectroscopy.
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氧化黄素、还原黄素和自由基黄素与分支酸合酶的结合。
DOI：
10.1021/bi951705u
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
R. Thorneley
中科院分区：
文献类型：
--
作者：
P. Macheroux;J. Petersen;S. Bornemann;D. Lowe;R. Thorneley
chismate合成酶（EC 4.6.1.4）在25℃下结合氧化核黄素-5′-磷酸单核苷酸（FMN）的KD为30微米，但在5-烯醇丙酮酰石草酸-3-磷酸（EPSP）存在时，KD降低到约20 nM。底物类似物(6R)-6-氟- epsp （KD = 36 nM）和choris酸盐（KD = 540 nM）也有类似的效果。氧化FMN的荧光在choris酸合酶的存在下被轻微猝灭。EPSP或(6R)-6-氟类似物的加入使荧光从520 nm移动到495nm。氯酸盐不会引起荧光发射最大值的偏移，而是使其猝灭。在EPSP、(6R)-6-氟EPSP或氯酸盐存在下，生成中性黄酮半醌。黄素自由基的电子顺磁共振（EPR）谱线宽度表明其为中性黄素半醌。自由基与(6R)-6-氟- epsp的冷冻溶液电子核双共振（ENDOR）显示出多个质子ENDOR线对。最大的分裂被分配到甲基β -质子的超精细偶联在异alloxazine环的位置8。超精细耦合（hfc）组件的值为A垂直= 8.07 MHz， A平行= 9.60 MHz，给出8.58 MHz的Aiso，与中性半醌形式一致。当氯代酸结合时，8-甲基质子与(6R)-6-氟- epsp的各向同性hfc偶联降低了约0.5 MHz，表明异alloxazine环体系内的自旋密度分布严重依赖于配体的性质。在氯酸合酶存在下，FMN的氧化还原电位比游离FMN （pH 7.0）的氧化还原电位高95 mV，相当于还原FMN的结合强度提高了1660倍。chis酸合酶结合的FMN的氧化还原电位的pH依赖性在pH 6和9之间表现出-30 mV / pH单位的斜率，表明黄素的双电子还原与一个质子的摄取有关；这一点，以及紫外可见光谱，与还原黄素以其单阴离子形式与choris酸合酶结合是一致的。
Chorismate synthase (EC 4.6.1.4) binds oxidized riboflavin-5'-phosphate mononucleotide (FMN) with a KD of 30 microM at 25 degrees C, but in the presence of 5-enolpyruvylshikimate-3-phosphate (EPSP), the KD decreases to ca. 20 nM. Similar effects occur with the substrate analogue (6R)-6-fluoro-EPSP (KD = 36 nM) and chorismate (KD = 540 nM). Fluorescence of oxidized FMN is slightly quenched in the presence of chorismate synthase. Addition of EPSP or the (6R)-6-fluoro analogue causes a shift of the fluorescence from 520 to 495 nm. Chorismate causes no shift in, but a quenching of, the fluorescence emission maximum. In the presence of EPSP, (6R)-6-fluoro-EPSP, or chorismate, the neutral flavinsemiquinone is generated. The electron paramagnetic resonance (EPR) line width of the flavin radical is indicative of a neutral flavinsemiquinone. Frozen solution electron nuclear double resonance (ENDOR) of the radical with (6R)-6-fluoro-EPSP shows a number of proton ENDOR line pairs. The largest splitting is assigned to a hyperfine coupling to the methyl group beta-protons at position 8 of the isoalloxazine ring. The hyperfine-coupling (hfc) components have values of A perpendicular = 8.07 MHz and A parallel = 9.60 MHz, giving Aiso of 8.58 MHz, consistent with a neutral semiquinone form. The isotropic hfc coupling of the 8-methyl protons with (6R)-6-fluoro-EPSP decreases by about 0.5 MHz when chorismate is bound, indicating that the spin density distribution within the isoalloxazine ring system depends critically on the nature of the ligand. The redox potential of FMN in the presence of chorismate synthase was 95 mV more positive than that of free FMN (at pH 7.0), equivalent to a 1660-fold tighter binding of reduced FMN. The pH dependence of the redox potential of chorismate synthase-bound FMN exhibits a slope of -30 mV per pH unit between pH 6 and 9, indicating that the two-electron reduction of the flavin is associated with the uptake of one proton; this, and the UV-visible spectrum, is consistent with the reduced flavin being bound to chorismate synthase in its monoanionic form.