Rapid determination of adenosine deaminase kinetics using fast-scan cyclic voltammetry.
Rapid determination of adenosine deaminase kinetics using fast-scan cyclic voltammetry.
复制标题
使用快速扫描循环伏安法快速测定腺苷脱氨酶动力学。
DOI：
10.1039/c0cp00294a
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
B. Venton
中科院分区：
文献类型：
--
作者：
Yida Xu;B. Venton
腺苷脱氨酶是一种参与嘌呤代谢的酶，其抑制剂被用作抗癌和抗病毒药物。在这项研究中，我们证明了碳纤维微电极上的快速扫描循环伏安法可以通过电化学监测腺苷浓度的降低来研究腺苷脱氨酶的动力学。缓冲液和盐浓度影响酶动力学和红-9-（2-羟基-3-nonyl）腺嘌呤（EHNA）和脱氧辅虫霉素（DCF）的抑制作用。在含有模拟脑脊液的盐的Tris缓冲液中，EHNA和DCF分别表现出非竞争性抑制，K(i)分别为1.7 +/- 0.6 nM和1.2 +/- 0.2 nM。然而，从Tris缓冲液中去除二价阳离子导致抑制具有竞争性，并将DCF的K(i)降低了两个数量级。在磷酸盐缓冲盐水中，EHNA的K(i)为1.0 +/- 0.2 nM， DCF的K(i)为3.6 +/- 0.3 pM，与文献值相似。腺苷脱氨酶也被AgNO(3)竞争性抑制，表明它对银毒性敏感。咖啡因被发现可以增加腺苷脱氨酶的活性。这是一种快速、简便的方法，用于筛选药物对酶动力学的影响，并可应用于其他酶促反应，其中反应物和产物的电活性有显著差异。
Adenosine deaminase is an enzyme involved in purine metabolism and its inhibitors are used as anticancer and antiviral drugs. In this study, we show that fast-scan cyclic voltammetry at carbon-fiber microelectrodes can be used to study the kinetics of adenosine deaminase by electrochemically monitoring decreases in adenosine concentration. Buffer and salt concentrations were shown to affect the enzyme kinetics and the inhibition by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin (DCF). In a Tris buffer containing salts that mimic cerebrospinal fluid, EHNA and DCF showed non-competitive inhibition with a K(i) of 1.7 +/- 0.6 nM and 1.2 +/- 0.2 nM, respectively. However, removing the divalent cations from the Tris buffer caused the inhibition to be competitive and reduced the K(i) for DCF by two orders of magnitude. In phosphate-buffered saline, the K(i) was 1.0 +/- 0.2 nM for EHNA and 3.6 +/- 0.3 pM for DCF, similar to literature values. Adenosine deaminase was also competitively inhibited by AgNO(3), showing it is susceptible to silver toxicity. Caffeine was found to increase adenosine deaminase activity. This is a fast, easy method for screening drug effects on enzyme kinetics and could be applied to other enzymatic reactions where there is a significant difference in the electroactivity of the reactant and product.
影响因子：
20.3
作者：
MURRAY, JL;LOFTIN, KC;HERSH, EM
通讯作者：
HERSH, EM
影响因子：
56.9
作者：
WILSON, DK;RUDOLPH, FB;QUIOCHO, FA
通讯作者：
QUIOCHO, FA