Humphreys Kristi J, Mirica Liviu M, Wang Yi, Klinman Judith P
Department of Chemistry, University of California, Berkeley, California 94720, USA.
J Am Chem Soc. 2009 Apr 8;131(13):4657-63. doi: 10.1021/ja807963e.
The mononuclear copper enzyme, galactose oxidase, has been investigated under steady-state conditions via O(2)-consumption assays using 1-O-methyl-alpha-D-galactopyranoside as the sugar substrate to produce an aldehyde at the C-6 position. The rate-determining step of the oxidative half-reaction was probed through the measurement of substrate and solvent deuterium and O-18 isotope effects on k(cat)/K(m)(O(2)). The reaction conforms to a ping-pong mechanism with the kinetic parameters for the reductive half, k(cat)/K(m)(S) = 8.3 x 10(3) M(-1) s(-1) at 10 degrees C and pH 7.0, comparing favorably to literature values. The oxidative half-reaction yielded a value of k(cat)/K(m)(O(2)) = 2.5 x 10(6) M(-1) s(-1). A substrate deuterium isotope effect of 32 was measured for the k(cat)/K(m)(S), while a smaller, but significant value of 1.6-1.9 was observed on k(cat)/K(m)(O(2)). O-18 isotope effects of 1.0185 with either protiated or deuterated sugar, together with the absence of any solvent isotope effect, lead to the conclusion that hydrogen atom transfer from reduced cofactor to a Cu(II)-superoxo intermediate is fully rate-determining for k(cat)/K(m)(O(2)). The measured O-18 isotope effects provide corroborative evidence for the reactive superoxo species in the dopamine beta-monooxygenase/peptidylglycine alpha-hydroxylating monooxygenase family, as well as providing a frame of reference for copper-superoxo reactivity. The combination of solvent and substrate deuterium isotope effects rules out solvent deuterium exchange into reduced enzyme as the origin of the relatively small substrate deuterium isotope effect on k(cat)/K(m)(O(2)). These data indicate fundamental differences in the hydrogen transfer step from the carbon of substrate vs the oxygen of reduced cofactor during the reductive and oxidative half-reactions of galactose oxidase.
通过以1-O-甲基-α-D-吡喃半乳糖苷作为糖底物在C-6位生成醛的耗氧测定法，在稳态条件下对单核铜酶——半乳糖氧化酶进行了研究。通过测量底物和溶剂的氘以及O-18同位素对k(cat)/K(m)(O(2))的影响，探究了氧化半反应的速率决定步骤。该反应符合乒乓机制，还原半反应的动力学参数k(cat)/K(m)(S)在10℃和pH 7.0时为8.3×10(3) M(-1) s(-1)，与文献值相比具有优势。氧化半反应得到的k(cat)/K(m)(O(2))值为2.5×10(6) M(-1) s(-1)。对于k(cat)/K(m)(S)，测得底物氘同位素效应为32，而对于k(cat)/K(m)(O(2))，观察到较小但显著的值为1.6 - 1.9。无论是质子化还是氘代糖的O-18同位素效应均为1.0185，同时不存在任何溶剂同位素效应，这导致得出结论：从还原辅因子到Cu(II)-超氧中间体的氢原子转移对于k(cat)/K(m)(O(2))是完全的速率决定因素。所测得的O-18同位素效应为多巴胺β-单加氧酶/肽基甘氨酸α-羟基化单加氧酶家族中的活性超氧物种提供了确证证据，同时也为铜-超氧反应性提供了参考框架。溶剂和底物氘同位素效应的组合排除了溶剂氘交换到还原酶中是对k(cat)/K(m)(O(2))相对较小的底物氘同位素效应的起源。这些数据表明在半乳糖氧化酶的还原和氧化半反应过程中，从底物的碳与还原辅因子的氧进行氢转移步骤存在根本差异。
