Rescue of the Orphan Enzyme Isoguanine Deaminase

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• 作者：Daniel S. Hitchcock ; Alexander A. Fedorov ; Elena V. Fedorov ; Lawrence J. Dangott ; Steven C. Almo ; Frank M. Raushel
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：June 28, 2011
• 年：2011
• 卷：50
• 期：25
• 页码：5555-5557
• 全文大小：687K
• 年卷期：v.50,no.25(June 28, 2011)
• ISSN：1520-4995

文摘

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k_cat = 49 s^鈥?, K_m = 72 渭M, and k_cat/K_m = 6.7 脳 10⁵ M^鈥? s^鈥?. The kinetic constants for the deamination of cytosine are as follows: k_cat = 45 s^鈥?, K_m = 302 渭M, and k_cat/K_m = 1.5 脳 10⁵ M^鈥? s^鈥?. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.