SPECTRAL AND KINETIC-STUDIES OF IMINE PRODUCT FORMATION IN THE OXIDATION OF P-(N,N-DIMETHYLAMINO)BENZYLAMINE ANALOGS BY MONOAMINE OXIDASE-B
SPECTRAL AND KINETIC-STUDIES OF IMINE PRODUCT FORMATION IN THE OXIDATION OF P-(N,N-DIMETHYLAMINO)BENZYLAMINE ANALOGS BY MONOAMINE OXIDASE-B
复制标题
DOI：
10.1021/bi00070a031
复制
发表时间：
1993-05-18
期刊：
影响因子：
2.9
通讯作者：
WALKER, MC
中科院分区：
文献类型：
--
作者：
EDMONDSON, DE;BHATTACHARYYA, AK;WALKER, MC
采用吸收光谱、稳态和停流动力学研究了牛肝单胺氧化酶B对-（N，N-二甲胺）和N-甲基-对-（N，N-二甲胺）苯胺的氧化脱胺反应。在中性pH值的转换实验中，用任何类似物观察到一种吸收中间体，其最大值为390 nm，并且由于质子化亚胺作为初始产物的形成而被确定。对-（N，N-二甲氨基）苯甲醛是由这两种底物类似物形成的最终产物。厌氧停流测量表明，N-甲基-p-（N，N-二甲胺）苄胺以1.8 s-1的极限速率还原酶结合黄素，同时由于质子化亚胺产物出现390 nm的吸收，极限速率为1.7 s-1。两种观察到的速率都略快于催化周转率（1.5 s-1）。在厌氧条件下，质子化N-甲基-对-（N，N-二甲氨基）苯亚胺的衰变比周转量慢得多（k = 4.8 x 10(4) s-1）。p-（N，N-二甲氨基）苄胺还原酶的极限速率为2.1 s-1，比催化转化率（1.2 s-1）快。在该底物中也观察到质子化亚胺的形成，其表观极限速率为1.3 s-1。在厌氧条件下，质子化对（N，N-二甲氨基）苯亚胺吸光度的衰减比催化转化率慢，但比醛生成速率快。在黄素还原和质子化亚胺生成过程中，氘的动力学同位素效应值均约为10。亚胺的衰变速率没有观察到同位素效应。这些结果表明，亚胺的质子化形式是单胺氧化酶b氧化苄胺的初始产物。还原后的酶-亚胺络合物对解离具有动力学稳定性，但与O2的反应速率与催化速率一致。从酶的氧化形式释放后，亚胺水解发生非酶性。
The oxidative deamination of p-(N,N-dimethylamino)benzylamine and N-methyl-p-(N,N-dimethylamino)benzylamine by bovine liver monoamine oxidase B has been investigated by absorption spectral, steady-state, and stopped-flow kinetic studies. An absorbing intermediate with a maximum at 390 nm is observed with either analogue in turnover experiments at neutral pH and is identified as due to the formation of protonated imine as the initial product. p-(N,N-Dimethylamino)benzaldehyde is the final product formed from either substrate analogue. Anaerobic stopped-flow measurements show N-methyl-p-(N,N-dimethylamino)benzylamine to reduce enzyme-bound flavin with a limiting rate of 1.8 s-1 concurrent with the appearance of a 390-nm absorption due to protonated imine product with a limiting rate of 1.7 s-1. Both observed rates are somewhat faster than catalytic turnover (1.5 s-1). Under anaerobic conditions, the decay of protonated N-methyl-p-(N,N-dimethylamino)benzenimine is much slower than turnover (k = 4.8 x 10(4) s-1). p-(N,N-Dimethylamino)benzylamine reduces the enzyme with a limiting rate of 2.1 s-1, which is faster than catalytic turnover (1.2 s-1). Protonated imine formation is also observed with this substrate with an apparent limiting rate of 1.3 s-1. The decay of the protonated p-(N,N-dimethylamino) benzenimine absorbance is slower than catalytic turnover but faster than the rate of aldehyde formation under anaerobic conditions. Deuterium kinetic isotope effect values of approximately 10 are observed both for flavin reduction and for protonated imine formation. No isotope effect is observed for the rate of imine decay. These results demonstrate that the protonated form of the imine is the initial product in the oxidation of benzylamines by monoamine oxidase B. The reduced enzyme-imine complex is kinetically stable to dissociation, but reacts with O2 at rates consistent with catalysis. Imine hydrolysis occurs nonenzymatically after release from the oxidized form of the enzyme.