Table 3 in How is the activity of shikimate dehydrogenase from the root of Petroselinum crispum (parsley) regulated and which side reactions are catalyzed?
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Table 3 Identification of SDH products by LC-MS analysis. List of products detected after 30 min incubation with (and without) SDH enzyme at 20 ◦ C. DHS, 3-dehydroshikimate; DHQ, 3-dehydroquinate; PCA, protocatechuic acid; SA, shikimic acid; QA, quinate. *the incubation time was 60 min at 20 ◦ C.SDH from P. crispum root (10 μl with specific activity 470 nmol.min -1.mg -1) was incubated in the reaction mixture (280 μl) containing 2 mM substrate (SA, DHS, DHQ, QA, GA, or PCA) and 0.14 mM cofactor (NADP, NADPH, NAD, NADH) in 100 mM Tris-HCl pH 8 (physiological direction) or 100 mM AMP-NaOH pH 9 at 20 ◦ C for 30 min. The reaction was initiated by adding SDH and stopped by adding 10 μl of 99 % (v/v) formic acid. Blank experiments were prepared similarly, albeit adding formic acid before the enzyme. Each determination was done at least 3-times, the average values and S. D. are shown. reactionproducts [nmol]SA+ NADPDHS: 5.7 ± 0.7PCA: 0.1 ± 0.0*DHS+ NADPHSA: 4.9 ± 0.2GA: 0.5 ± 0.0PCA: 0.7 ± 0.1DHQ: 1.1 ± 0.0DHS+ NADPGA: 0.1 ± 0.0PCA: 0.3 ± 0.2DHS+ NADPGA: 2.1 ± 0.4– without SDHPCA: 2.7 ± 0.6DHQ+ NADPDHS: 17.4 ± 1.4PCA: 4.9 ± 0.4*DHQ+ NADPHDHS: 3.4 ± 0.8SA: 1.4 ± 0.2GA: 1.1 ± 0.5PCA: 1.3 ± 0.2DHQ+ NADHNo products detected.QA+ NADNo products detected.PCA+ NADPHNo products detected.
表3 液相色谱-质谱（LC-MS）分析鉴定莽草酸脱氢酶（SDH）产物。20℃下与SDH酶（设置有无酶两组对照）孵育30分钟后检测到的产物列表。缩写说明：DHS为3-脱氢莽草酸（3-dehydroshikimate）；DHQ为3-脱氢奎尼酸（3-dehydroquinate）；PCA为原儿茶酸（protocatechuic acid）；SA为莽草酸（shikimic acid）；QA为奎尼酸（quinate）。注：* 对应孵育时间为60分钟，温度20℃。 取欧芹（P. crispum）根源SDH（10 μl，比活为470 nmol·min⁻¹·mg⁻¹），加入至280 μl反应体系中，该体系包含2 mM底物（SA、DHS、DHQ、QA、GA及PCA）与0.14 mM辅因子（烟酰胺腺嘌呤二核苷酸磷酸（NADP）、烟酰胺腺嘌呤二核苷酸磷酸还原型（NADPH）、烟酰胺腺嘌呤二核苷酸（NAD）、烟酰胺腺嘌呤二核苷酸还原型（NADH）），反应缓冲液分别为100 mM 三羟甲基氨基甲烷盐酸盐（Tris-HCl，pH 8.0，模拟生理反应方向）或100 mM 腺苷一磷酸氢氧化钠（AMP-NaOH，pH 9.0），于20℃孵育30分钟。反应通过加入SDH启动，通过添加10 μl 99%（体积分数）甲酸终止。空白对照实验操作流程一致，仅需提前加入甲酸以预先灭活酶活。每组实验至少重复3次，结果以平均值±标准偏差（S.D.）呈现。 反应产物 单位：nmol SA + NADP： DHS：5.7 ± 0.7 PCA：0.1 ± 0.0* DHS + NADPH： SA：4.9 ± 0.2 GA：0.5 ± 0.0 PCA：0.7 ± 0.1 DHQ：1.1 ± 0.0 DHS + NADP： GA：0.1 ± 0.0 PCA：0.3 ± 0.2 DHS + NAD： GA：2.1 ± 0.4 —— 无SDH组： PCA：2.7 ± 0.6 DHQ + NADP： DHS：17.4 ± 1.4 PCA：4.9 ± 0.4* DHQ + NADPH： DHS：3.4 ± 0.8 SA：1.4 ± 0.2 GA：1.1 ± 0.5 PCA：1.3 ± 0.2 DHQ + NADH：未检测到产物 QA + NAD：未检测到产物 PCA + NADPH：未检测到产物
