pH and kinetic isotope effects in D-amino acid oxidase catalysis - Evidence for a concerted mechanism in substrate dehydrogenation via hydride transfer
pH and kinetic isotope effects in D-amino acid oxidase catalysis - Evidence for a concerted mechanism in substrate dehydrogenation via hydride transfer
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DOI：
10.1046/j.1432-1033.2001.02462.x
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发表时间：
2001-11-01
期刊：
影响因子：
--
通讯作者：
Ghisla, S
中科院分区：
文献类型：
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作者：
Harris, CM;Pollegioni, L;Ghisla, S
研究了pH、溶剂同位素和一次同位素置换对红酵母d -氨基酸氧化酶对底物脱氢的影响。d -丙氨酸还原酶- fad的速率常数随pH值增加近四倍，反映表观pK(a)值接近6和接近8，并在高和低pH下达到平台。在使用d -丙氨酸和d -天冬酰胺作为底物的所有预稳态和稳态动力学实验中都观察到这种曲线，并且与催化必需的碱的操作不一致。在pH为6时，与d -丙氨酸反应的溶剂氘同位素效应为3.1 +/- 1.1；pH值为10时降至1.2 +/- 0.2。[2-D] d -丙氨酸对还原速率的主要底物同位素效应在低pH下为9.1 +/- 1.5，在高pH下为2.3 +/- 0.3，在pH 6.0时，[2-D] d -丙氨酸的溶剂同位素效应为2.9 +/- 0.8，在D2O中，主要同位素效应为8.4 +/- 2.4。因此，原生和溶剂动力学同位素效应（KIEs）与其他同位素的存在无关，即“双”动力学同位素效应是单个KIEs的产物，与底物与氢的两个键的断裂一致的过渡状态相一致。这些结果支持了d -氨基酸氧化酶脱氢反应的氢化物转移机制，并反对在该过程中存在任何中间体。近似于8的pK（A,app）被解释为由底物氨基酸α -氨基的微观电离引起，但也包括动力学参数的贡献。
The effects of pH, solvent isotope, and primary isotope replacement on substrate dehydrogenation by Rhodotorula gracilis D-amino acid oxidase, were investigated. The rate constant for enzyme-FAD reduction by D-alanine increases approximate to fourfold with pH, reflecting apparent pK(a) values of approximate to 6 and approximate to 8, and reaches plateaus at high and low pH. Such profiles are observed in all presteady-state and steady-state kinetic experiments, using both D-alanine, and D-asparagine as substrates, and are inconsistent with the operation of a base essential to catalysis. A solvent deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with D-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary substrate, isotope effect on the reduction rate with [2-D]D-alanine is 9.1 +/- 1.5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with, [2-D]D-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) are independent of the presence of the other isotope, i.e. the 'double' kinetic isotope effect is the product of the individual KIEs, consistent with a transition state in which rapture of the two bonds of the substrate to hydrogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in D-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pK(a,app) of approximate to 8 is interpreted to arise from the microscopic ionization of the substrate amino acid alpha -amino group, but also includes contributions from kinetic parameters.