Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Avenue, 48201, Detroit, MI, USA.
Chembiochem. 2023 Sep 15;24(18):e202300347. doi: 10.1002/cbic.202300347. Epub 2023 Aug 16.
The enzyme N -carboxylaminoinidazole ribonucleotide (N -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N -CAIR synthetase. During our research on identifying inhibitors of N -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N -CAIR mutase.
酶 N -羧基亚氨基咪唑核苷酸（N -CAIR）变位酶存在于微生物从头嘌呤生物合成中，但在人类中不存在，因此成为有吸引力的抗菌靶标。N -CAIR 变位酶催化 N -CAIR 合成羧基氨基咪唑核苷酸（CAIR），N -CAIR 本身由酶 N -CAIR 合成酶从氨基咪唑核苷酸（AIR）制备。在研究鉴定 N -CAIR 变位酶抑制剂的过程中，我们开发了一种创新的荧光测定法来测量该酶的活性。该测定法依赖于我们最近偶然观察到的 AIR 与靛红可逆反应。荧光标记的靛红与 AIR 的反应导致荧光强度大幅增加，从而可以测量溶液中 AIR 的浓度。从这个观察结果中，我们开发了一种可重复的、非连续的测定法，该测定法可以复制酶的已知动力学参数，并可以轻松检测到公认的酶抑制剂。该测定法应该在筛选针对 N -CAIR 变位酶的抑制剂方面具有实用价值。
