Rudjer Bošković Institute, Department for Physical Chemistry , Zagreb , Croatia.
J Enzyme Inhib Med Chem. 2013 Oct;28(5):1094-104. doi: 10.3109/14756366.2012.716834. Epub 2012 Sep 20.
Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E•I complex and first-order rate constant for inactivation were determined by incubation (Ki* = 26.6 ± 2.8 µM, k2 = 12.2 ± 0.6 min-1) or progress curve (Ki* = 6.5 ± 1.5 µM, k2 = 0.11 ± 0.01 min-1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t1/2 = 11.3 ± 0.2 h), matrix-assisted laser desorption/ionization (MALDI, t1/2 = 13.5 ± 0.4 h), and electro-spray ionization (ESI, t1/2 = 12.2 ± 0.5 h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.
首次采用丝氨酸蛋白酶抑制剂 3,4-二氯异香豆素（DCI），通过活性测量和质谱（MS）对脂肪酶抑制作用进行了动力学特征分析。DCI 对 SGNH 超家族成员的里氏木霉胞外脂肪酶（SrLip）的抑制遵循两步不可逆抑制机制。通过孵育（Ki* = 26.6 ± 2.8 μM，k2 = 12.2 ± 0.6 min-1）或进展曲线（Ki* = 6.5 ± 1.5 μM，k2 = 0.11 ± 0.01 min-1）方法确定非共价 E•I 复合物的离解常数和失活的一级速率常数。通过活性测量（t1/2 = 11.3 ± 0.2 h）、基质辅助激光解吸/电离（MALDI，t1/2 = 13.5 ± 0.4 h）和电喷雾电离（ESI，t1/2 = 12.2 ± 0.5 h）MS 确定用 10 倍摩尔过量 DCI 抑制的脂肪酶的复活动力学半衰期。通过在酶与 DCI 的近等摩尔浓度下孵育，然后进行活性和 MS 测量来确定活性 SrLip 浓度。
