Stereoselectivity in the enzymic oxidation and nonenzymic hydrogen-exchange reactions of dihydroorotate
Stereoselectivity in the enzymic oxidation and nonenzymic hydrogen-exchange reactions of dihydroorotate
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二氢乳清酸酶氧化和非酶氢交换反应中的立体选择性
DOI：
10.1021/ja00288a036
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发表时间：
1985
影响因子：
15
通讯作者：
M. Johnston
中科院分区：
文献类型：
--
作者：
L. Keys;M. Johnston
(S)-5 6-二氢乳清酸（1）在氧化氘缓冲液（100 mMNaP 3，pD7.8，37 ℃）中与溶剂氘进行立体选择性氢交换反应。交换的高场1H NMR分析揭示了（5/？，6S）-[5- 2 H]二氢乳清酸盐（3）在表观一级反应中（tcobsd = 5.4 × 10 - 3 h-137 ℃）。还观察到氘掺入到1的5-pro-S位置;然而，pro-S交换比pro-R交换慢得多（kobsi= 4.8 × 10 - 4 h-1，37 ℃）。还观察到（S）-二氢乳清酸甲酯（6）和6-甲基-（R，5）-二氢尿嘧啶（7）的立体选择性交换，尽管这些反应仅具有适度的立体参照性。我们已经利用了5-pro-R氢交换的这种立体化学偏好，通过制备两种富氘二氢乳清酸盐的非对映异构体3（从D2 Q缓冲液中的1）和5（从H2O缓冲液中的（S）-[5，5- 2 H2]二氢乳清酸盐4）。这些手性氘代二氢乳清酸酯用于阐明由牛肝线粒体二氢乳清酸脱氢酶（EC 1.3. 3.1）。酶促氧化容易遵循高场NMR光谱;数据表明，酶是绝对立体选择性的非对映异构体C5中心。在二氢乳清酸盐转化为乳清酸盐的过程中，只有前S氢（或氘）从C5中损失，从而描述了底物的反式氧化。因此，我们报告了一个有趣的观察结果，即二氢乳清酸（1-* 2）的酶促加工在C5处显示出与非酶促C5-氢交换（1-* 3）相反的立体选择性。这些结果进行检查参考构象，立体电子，和静电的因素，可能会影响酶催化和非酶反应的立体化学过程。
(S)-5, 6-Dihydroorotate (1) undergoes a stereoselective hydrogen-exchange reaction with solvent deuterons in deuterium oxide buffer (100 mM NaP¡,'pD 7.8, 37 C). High-field* H NMR analysis of the exchange reveals the formationof (5/?, 6S)-[5-2H] dihydroorotate (3) in an apparent first-order process (/cobsd= 5.4 X 10" 3 h~\37 C). Deuterium incorporation into the 5-pro-S position of 1 is alsoobserved; pro-S exchange, however, occurs considerably more slowly (kobsi= 4.8 X 10" 4 h'1, 37 C) than does pro-R exchange. Stereoselective exchange was also seen for methyl (S)-dihydroorotate (6) and for 6-methyl-(R, 5)-dihydrouracil (7), although these reactions are only modestly stereopreferential. We have capitalized on this stereochemical preference for 5-pro-R hydrogen exchange by preparing two diastereomers of deuterium-enriched dihydroorotates, 3 (from 1 in D2Q buffer) and 5 (from (S)-[5, 5-2H2] dihydroorotate 4 in H20 buffer). These chirally deuterated dihydroorotates were used to elucidate the stereochemical course of the reaction (1—» 2) catalyzed by the beef liver mitochondrial dihydroorotate dehydrogenase (EC 1.3. 3.1). Enzymatic oxidation was readily followed by high-field NMR spectrometry; the data show that the enzyme is absolutely stereoselective with reference to the diastereotopic C5 center. Only the pro-S hydrogen (or deuteron) is lost from C5 during conversion of dihydroorotate to orotate, thereby describing a trans oxidationof substrate. Thus, we report the interesting observation that enzymatic processing of dihydroorotate (1—* 2) shows stereoselectivity at C5 opposite to that of the nonenzymatic C5-hydrogen exchange (1-* 3). These results are examined with reference to the conformational, stereoelectronic, and electrostatic factors that may influence the stereochemical course of both the enzyme-catalyzed and nonenzymatic reactions.