Niaura Gediminas, Kuprionis Zenonas, Ignatjev Ilja, Kazemekaite Maryte, Valincius Gintaras, Talaikyte Zita, Razumas Valdemaras, Svendsen Allan
Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania, EKSPLA Ltd., Savanoriu Av. 231, LT-02300 Vilnius, Lithuania.
J Phys Chem B. 2008 Apr 3;112(13):4094-101. doi: 10.1021/jp075950m. Epub 2008 Mar 7.
The infrared-visible sum-frequency generation (SFG) vibrational spectroscopy was used to probe enzymatic activity of Thermomyces lanuginosus lipase (TLL) at air/water interface. A monolayer of amphiphilic O-palmitoyl-2,3-dicyanohydroquinone (PDCHQ), containing target ester group and two CN groups serving as vibrational markers, was utilized as an enzyme substrate. SFG data revealed the detailed molecular scale structure and properties of the PDCHQ layer at the interface. In particular, we demonstrate that hydrophilic headgroup of PDCHQ is mainly in the form of an oxyanion, and the enzyme-induced cleavage of the ester bond could be spectroscopically monitored by the disappearance of the intense C tripple bond N resonance at 2224 cm(-1). The enzymatic nature of the ester bond cleavage was confirmed by the control experiments with deactivated S146A mutant variant of TLL. By comparing action of wild type (WT) TLL and its inactive S146A mutant, it was shown that two effects take place at the interface: disordering of the lipid monolayer due to the adsorption of enzyme and enzymatic cleavage of the ester bond. The concentration of enzyme as low as 10 nM could be easily sensed by the SFG spectroscopy. We present spectroscopic evidence that upon hydrolysis one of the products, 2,3-dicyanohydroquinone, leaves the surface, while the other, palmitic acid, remains at air/water interface in predominantly undissociated form with the mono-hydrogen-bonded carbonyl group. Strong amide I (1662 cm(-1)) and amide A (3320 cm(-1)) SFG signals from TLL suggest that enzyme molecules position themselves at air/water interface in an orderly fashion. Presented work demonstrates the potential of SFG spectroscopy for in situ real-time monitoring of enzymatic processes at air/water interface.
利用红外 - 可见和频振动光谱法探测嗜热栖热菌脂肪酶（TLL）在空气/水界面的酶活性。以含有目标酯基和两个作为振动标记物的氰基的两亲性O - 棕榈酰 - 2,3 - 二氰基对苯二酚（PDCHQ）单分子层作为酶底物。和频光谱数据揭示了界面处PDCHQ层的详细分子尺度结构和性质。特别地，我们证明PDCHQ的亲水头基主要以氧阴离子形式存在，并且酯键的酶促裂解可以通过2224 cm(-1)处强烈的C≡N共振消失进行光谱监测。通过对失活的TLL S146A突变变体进行对照实验，证实了酯键裂解的酶促性质。通过比较野生型（WT）TLL及其无活性的S146A突变体的作用，表明在界面处发生了两种效应：由于酶的吸附导致脂质单分子层无序化以及酯键的酶促裂解。和频光谱法能够轻松检测低至10 nM的酶浓度。我们提供了光谱证据表明，水解后其中一种产物2,3 - 二氰基对苯二酚离开表面，而另一种产物棕榈酸则以主要未离解的形式保留在空气/水界面，其羰基形成单氢键。来自TLL的强酰胺I（1662 cm(-1)）和酰胺A（3320 cm(-1)）和频光谱信号表明酶分子以有序方式定位在空气/水界面。目前的工作证明了和频光谱法在原位实时监测空气/水界面酶促过程方面的潜力。
