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首页> 美国卫生研究院文献>other >A Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in Formation of the Oxygen-Reactive Complex
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A Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in Formation of the Oxygen-Reactive Complex

机译：酪氨酸羟化酶一氧化氮配合物中氨基酸和四氢蝶呤结合的脉冲EPR研究：氧反应性配合物形成中底物重排的证据。

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Tyrosine hydroxylase is a non-heme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin and molecular oxygen. We have used nitric oxide as an O2 surrogate to poise Fe(II) at the catalytic site in an S=3/2, {FeNO}7 form amenable to EPR spectroscopy. 2H-Electron Spin Echo Envelope Modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}7 paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}7 center to the closest deuteron on 6,7-2H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-2H-tyrosine resulted in reorientation of the magnetic axes of the S=3/2 {FeNO}7 center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase.
机译：酪氨酸羟化酶是神经系统中发现的一种非血红素铁酶，可催化酪氨酸的羟化反应以形成L-3,4-二羟基苯丙氨酸，这是儿茶酚胺神经递质生物合成中的限速步骤。催化需要结合三种底物：酪氨酸，四氢生物蝶呤和分子氧。我们使用一氧化氮作为O2的替代物，以E =光谱的S = 3/2 {FeNO} 7 形式在催化位上保持Fe（II）的平衡。然后使用 2 H电子自旋回波包络调制来测量特定氘代底物酪氨酸和辅因子6-甲基四氢蝶呤相对于{FeNO} 7 < / sup>顺磁性中心。我们的结果表明，酪氨酸的添加会触发酶的构象变化，从而缩短从{FeNO} 7 中心到最近的氘代6,7- 2 的距离。 H-6-甲基四氢蝶呤从> 5.9到4.4±0.2Å。相反，在3,5- 2 H-酪氨酸处理的酶样品中添加6-甲基四氢蝶呤会导致S = 3/2 {FeNO} 7 <相对于氘代基板居中。两者合计，这些结果表明，底物和辅因子的配位将NO的配位引导到活性部位的Fe（II）。并行研究未偶联的酪氨酸羟化酶变体E332A的季铵盐配合物，与底物酪氨酸和辅因子6-甲基四氢蝶呤的超精细偶联没有变化。我们的结果是在先前对酪氨酸羟化酶和苯丙氨酸羟化酶进行的光谱和X射线晶体学研究的背景下讨论的。

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