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首页> 外文期刊>Biochemistry >Relationship of stopped flow to steady state parameters in the dimeric copper amine oxidase from Hansenula polymorpha and the role of zinc in inhibiting activity at alternate copper-containing subunits
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Relationship of stopped flow to steady state parameters in the dimeric copper amine oxidase from Hansenula polymorpha and the role of zinc in inhibiting activity at alternate copper-containing subunits

机译：多形汉逊酵母中二聚体铜胺氧化酶中的稳定流与稳态参数的关系以及锌在抑制含铜亚基的活性中的作用

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摘要

The expression of a copper amine oxidase (CAO) from Hansenula polymorpha in Saccharomyces cerevisiae under differing culture conditions leads to the incorporation of varied levels of CAO-bound zinc. The presence of substantial amount of zinc results in two distinctive enzyme species, designated as the fast and slow enzymes. Both forms are rapidly reduced by substrate methylamine with a rate constant of 199 s(-1) but behave remarkably differently in their oxidation rates; the fast enzyme is oxidized by dioxygen at a rate of 22.1 s(-1), whereas the slow enzyme reacts at a rate of 1.8x10(-4) s(-1). The apparent k(cat) of the enzyme preparation is linearly proportional to the fraction of the fast enzyme, with an extrapolated value of 6.17 s(-1) when the enzyme is 100% in its "fast" form. A comparison of rate constants for cofactor reduction and reoxidation steps, measured in stopped flow experiments, to the extrapolated k(cat) implicates additional steps in the steady state reaction. Measurement of the proportion of oxidized (ETPQ(ox)) and reduced cofactor (ETPQ(red)) under steady state conditions indicates approximately 50% of each cofactor form at 0.8 or 2 mM methylamine. Kinetic isotope effect measurements using deuterated amine substrate lead to the following steady state values: (D)(k(red))=8.5 (0.5), (D)(k(cat))=1.7 (0.1), and (D)(k(cat)/K-m)=4.3 (0.2). The collective data allow the calculation of partially rate-determining constants during the reductive half-reaction (ca. 200 s(-1) for binding of substrate to ETPQ(ox) and 27.9 s(-1) for release of aldehyde product or a protein isomerization from ETPQ(red)); an additional step with a rate constant of 13.2 s(-1) is assigned to the oxidative half-reaction, most likely for the release of product hydrogen peroxide. These results, together with the sole detection of oxidized and reduced cofactor during rapid scanning stopped flow experiments, indicate that four steps contribute to k(cat), with the first electron transfer from cofactor to O-2 contributing ca. 29%. An investigation of the relationship between the copper content and the extent of the fast enzyme shows that only the copper-containing homodimer is capable of rapid reoxidation and that zinc-copper heterodimers are incapable of rapid turnover at either subunit. This implies communication between the metal sites of the two subunits per dimer that impacts O-2 binding and/or electron transfer from reduced cofactor to bound O-2.
机译：在不同的培养条件下，酿酒酵母中的多形汉逊酵母中的铜胺氧化酶（CAO）的表达导致掺入不同水平的CAO结合锌。大量锌的存在会导致两种独特的酶，分别称为快酶和慢酶。两种形式均被底物甲胺迅速还原，速率常数为199 s（-1），但其氧化速率表现出明显不同。快速酶被双氧以22.1 s（-1）的速度氧化，而慢速酶以1.8x10（-4）s（-1）的速度进行反应。酶制剂的表观k（cat）与快速酶的比例成线性比例，当酶以“快速”形式为100％时，外推值为6.17 s（-1）。在停止流动实验中测得的辅因子还原和再氧化步骤的速率常数与外推k（cat）的比较暗示了稳态反应中的其他步骤。在稳态条件下对氧化的（ETPQ（ox））和还原的辅因子（ETPQ（red））的比例进行测量表明，在0.8或2 mM的甲胺中，每种辅因子形式约占50％。使用氘化胺底物进行的动力学同位素效应测量得出以下稳态值：（D）（k（红色））= 8.5（0.5），（D）（k（cat））= 1.7（0.1）和（D） （k（cat）/ Km）= 4.3（0.2）。收集的数据可用于计算还原半反应期间的部分速率决定常数（对于底物与ETPQ（ox）的结合，约为200 s（-1）;对于醛产物或乙醛的释放，约为27.9 s（-1）。来自ETPQ（红色）的蛋白质异构化;速率常数为13.2 s（-1）的附加步骤分配给了氧化半反应，最有可能释放出产物过氧化氢。这些结果，以及在快速扫描停止流动实验期间仅检测到氧化和还原的辅因子的过程，表明四个步骤导致了k（cat），第一次电子从辅因子转移到O-2大约贡献了k.cat。 29％。对铜含量和快速酶程度之间关系的研究表明，仅含铜的同型二聚体能够快速再氧化，而锌-铜异二聚体在任一亚基上均不能快速转换。这意味着每个二聚体两个亚基的金属位点之间的连通会影响O-2的结合和/或电子从还原的辅因子到结合的O-2的转移。

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