He Jason Z, Lu Yuqing, Jain Neha, Churchill David G, Wong Lu Shin
Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
Department of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, U.K.
ACS Omega. 2024 Jun 25;9(27):29939-29946. doi: 10.1021/acsomega.4c05409. eCollection 2024 Jul 9.
This study reports the development of fluorometric assays for the detection and quantification of silyl hydrolase activity using silicatein as a model enzyme. These assays employed a series of organosilane substrates containing either mycophenolate or umbelliferone moieties, which become fluorescent upon hydrolysis of a scissile Si-O bond. Among these substrates, the mycophenolate-derived molecule MycoF, emerged as the most promising candidate due to its relative stability in aqueous media, which resulted in good differentiation between the enzyme-catalyzed and uncatalyzed background hydrolysis. The utility of MycoF was also demonstrated in the detection of enzyme activity in cell lysates and was found to be capable of qualitative identification of positive "hit" candidates in a high-throughput format. These fluorogenic substrates were also suitable for use in quantitative kinetic assays, as demonstrated by the acquisition of their Michaelis-Menten parameters.
本研究报告了以硅酸酶为模型酶，开发用于检测和定量硅烷水解酶活性的荧光测定法。这些测定法使用了一系列含有霉酚酸或伞形酮部分的有机硅烷底物，这些底物在可裂解的Si-O键水解后会发出荧光。在这些底物中，源自霉酚酸的分子MycoF因其在水性介质中的相对稳定性而成为最有前景的候选物，这导致酶催化水解和未催化背景水解之间有良好的区分度。MycoF在细胞裂解物中酶活性检测中的实用性也得到了证明，并且发现它能够以高通量形式对阳性“命中”候选物进行定性鉴定。如通过获取它们的米氏参数所证明的，这些荧光底物也适用于定量动力学测定。
