Interaction of non-conjugated olefinic substrate analogues with dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.
Interaction of non-conjugated olefinic substrate analogues with dopamine beta-monooxygenase: catalysis and mechanism-based inhibition.
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非共轭烯属底物类似物与多巴胺β-单加氧酶的相互作用：催化和基于机制的抑制。
DOI：
10.1042/bj3060077
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
May,SW
中科院分区：
文献类型：
--
作者：
Sirimanne,SR;May,SW
多巴胺β-单加氧酶(DBM；EC 1.14.17.1)与典型的非共轭烯烃底物2-(1-环己烯基)乙胺(CyHEA)的反应[见Sirimanne和May(1988)J.Am化学。SoC。110,7560-7561]，对其进行了表征。环己烷在DBM催化下发生简单的烯丙基羟基化反应生成(R)-2-氨基-1-(1-环己烯基)乙醇(CyHEA-OH)，没有检测到环氧化或烯丙基羟化反应，生成(R)-2-氨基-1-(1-环己烯基)乙醇(CyHEA-OH)，没有环氧化或烯丙基重排反应，立体化学与DBM催化的苄基羟基化和硫氧化反应一致。那只猫。S-1对环己烷的氧合作用约为kCAT的75%。对于酪胺，通常用于DBM活性测定的底物。DBM催化的CyHEA氧化也导致DBM基于机理的失活，失活反应产生KACT。=0.3min-1，pH 5.0，37℃，分配比16,000。虽然CyHEA周转和失活都表现出正常的动力学，但CyHEA处理也会导致DBM中铜的逐渐耗尽；然而，当分析溶液中存在足够的铜时，基于机理的不可逆DBM失活发生与这种铜耗尽无关。CyHEA依赖周转的DBM失活的一种可能机制涉及最初提取烯丙基氢以形成共振稳定的烯丙基自由基，然后该自由基可以分割成产物或遭受活性中心残基的攻击。非环、非共轭的烯烃类似物对DBM的底物活性降低。因此，kcat。对于顺式-2-己烯胺的氧化，也只产生烯丙醇产品，仅为环己烷的14%。类似地，2-氨基甲基-1-戊烯对翻转相关失活的Kinact./Ki比苯基烯烃小一个数量级以上。我们的结果表明，DBM催化了许多非共轭烯烃底物类似物的烯丙基氧化，既没有发生环氧化反应，也没有发生烯丙基重排反应。没有来自非共轭烯烃底物的环氧化物产物意味着DBM的活性铜氧物种不能影响非共轭烯烃部分形成自由基阳离子。DBM和细胞色素P-450之间的鲜明对比可能反映了这两种酶作用的活性氧物种氧化还原电位的差异。
The reaction of dopamine beta-monooxygenase (DBM; EC 1.14.17.1) with the prototypical non-conjugated olefinic substrate, 2-(1-cyclohexenyl)ethylamine (CyHEA) [see Sirimanne and May (1988) J. Am. Chem. Soc. 110, 7560-7561], was characterized. CyHEA undergoes facile DBM-catalysed allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic hydroxylation to form (R)-2-amino-1-(1-cyclohexenyl)ethanol (CyHEA-OH) without detectable epoxidation or allylic rearrangement, and with stereochemistry consistent with that of DBM-catalysed benzylic hydroxylation and sulphoxidation. The kcat. of 90 s-1 for CyHEA oxygenation is about 75% of the kcat. for tyramine, the substrate commonly used in assays of DBM activity. DBM-catalysed oxygenation of CyHEA also results in mechanism-based inactivation of DBM, with the inactivation reaction yielding kinact. = 0.3 min-1 at pH 5.0 and 37 degrees C, and a partition ratio of 16,000. Although both CyHEA turnover and inactivation exhibit normal kinetics, CyHEA processing also results in gradual depletion of copper from DBM; however, mechanism-based irreversible DBM inactivation occurs independent of this copper depletion when sufficient copper is present in the assay solution. A likely mechanism for turnover-dependent DBM inactivation by CyHEA involves initial abstraction of an allylic hydrogen to form a resonance-stabilized allylic radical, which can then either partition to product or undergo attack by an active-site residue. Acyclic, non-conjugated olefinic analogues exhibit diminished substrate activity toward DBM. Thus, kcat. for oxygenation of cis-2-hexenylamine, which also produces only allylic alcohol product, is only 14% of that for CyHEA. Similarly, kinact./KI for turnover-dependent inactivation by the acyclic olefin 2-aminomethyl-1-pentene is more than an order of magnitude smaller than that for benzylic olefins. Our results establish that DBM catalyses allylic oxygenation of a number of non-conjugated olefinic substrate analogues with neither epoxidation nor allylic rearrangement occurring. The absence of epoxide products from non-conjugated olefinic substrates implies an inability of the activated copper-oxygen species of DBM to effect radical cation formation from a non-conjugated olefinic moiety. The striking contrast between DBM and cytochrome P-450, which carries out both epoxidation and allylic oxidation with non-conjugated olefinic substrates, is probably a reflection of the differences in redox potential of the activated oxygen species operative for these two enzymes.