Kozlova N O, Bruskovskaya I B, Melik-Nubarov N S, Yaroslavov A A, Kabanov V A
Department of Polymer Science, School of Chemistry, Moscow State University, Leninskiye Gory, Moscow, Russian Federation.
FEBS Lett. 1999 Nov 19;461(3):141-4. doi: 10.1016/s0014-5793(99)01449-0.
A set of artificially hydrophobized alpha-chymotrypsin derivatives, carrying 2-11 stearoyl residues per enzyme molecule, were synthesized and their catalytic parameters and conformation in water solution and in the liposome-bound state were investigated. Hydrophobization of alpha-chymotrypsin and its further incorporation into phosphatidylcholine (PC) liposomes have no effect on the rate constant of the N-acetyl-L-tyrosine ethyl ester (ATEE) ester bond hydrolysis (k(cat)). At the same time, an increase in the number of stearoyl residues attached to the enzyme results in a drastic decrease of ATEE binding to the active center (K(M) increase). Incorporation of the hydrophobized enzyme into the PC liposome membrane results in K(M) recovery to nearly that of native alpha-chymotrypsin. The above changes are accompanied by partial unfolding of the enzyme molecules observed by fluorescence measurements. The obtained results are of interest to mimic the contribution of surface hydrophobic sites in the functioning of membrane proteins.
合成了一组人工疏水化的α-胰凝乳蛋白酶衍生物，每个酶分子带有2 - 11个硬脂酰残基，并研究了它们在水溶液和脂质体结合状态下的催化参数和构象。α-胰凝乳蛋白酶的疏水化及其进一步掺入磷脂酰胆碱（PC）脂质体对N-乙酰-L-酪氨酸乙酯（ATEE）酯键水解的速率常数（k(cat)）没有影响。同时，连接到酶上的硬脂酰残基数量增加导致ATEE与活性中心的结合急剧减少（K(M)增加）。将疏水化酶掺入PC脂质体膜导致K(M)恢复到接近天然α-胰凝乳蛋白酶的水平。上述变化伴随着通过荧光测量观察到的酶分子的部分展开。所得结果对于模拟表面疏水位点在膜蛋白功能中的作用具有重要意义。
