Beld Joris, Cang Hu, Burkart Michael D
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358 (USA).
Angew Chem Int Ed Engl. 2014 Dec 22;53(52):14456-61. doi: 10.1002/anie.201408576. Epub 2014 Oct 29.
The acyl carrier protein (ACP) from fatty acid synthases sequesters elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. We exploited solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. The addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, thus enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity was confirmed by the use of a dual solvatochromic cross-linking probe and solution-phase NMR spectroscopy. The chain-flipping mechanism was visualized by single-molecule fluorescence techniques, thus demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII.
脂肪酸合酶中的酰基载体蛋白（ACP）将延长产物隔离在其疏水核心内，但这种动态机制仍知之甚少。我们利用了连接到ACP上的溶剂化显色泛酰巯基乙胺探针，该探针在被隔离时会发出荧光。添加催化伴侣会将货物从ACP中吸引出来并进入酶的活性位点，从而增强荧光以揭示难以捉摸的链翻转机制。通过使用双溶剂化显色交联探针和溶液相核磁共振光谱法证实了这种活性。通过单分子荧光技术观察到了链翻转机制，从而证明了大肠杆菌ACP与其酮酰基合酶催化伴侣KASII之间的特异性。
