One-Pot and Sequential Organic Chemistry on an Enzyme Surface to Tether a Fluorescent Probe at the Proximity of the Active Site with Restoring Enzyme Activity

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• 作者：Yousuke Takaoka ; Hiroshi Tsutsumi ; Noriyuki Kasagi ; Eiji Nakata ; and Itaru Hamachi
• 刊名：Journal of the American Chemical Society
• 出版年：2006
• 出版时间：March 15, 2006
• 年：2006
• 卷：128
• 期：10
• 页码：3273 - 3280
• 全文大小：327K
• 年卷期：v.128,no.10(March 15, 2006)
• ISSN：1520-5126

文摘

A new and simple method to tether a functional molecule at the proximity of the active site of anenzyme has been successfully developed without any activity loss. The one-pot sequential reaction wasconducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and thesubsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in theoverall steps under mild conditions. The enzymatic activity assay demonstrated that the release of theaffinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxyderivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeledhCAII. Such restoring of the activity upon the sequential modification is quite unique compared toconventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reactionwas selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. Whenthe fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can actas a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of thismethod, that is (i) the modification is directed to the proximity of the active site and (ii) the sequentialreaction re-opens the active site cavity of the target enzyme.