van Kuiken B A, Behnke W D
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH 45267.
Biochim Biophys Acta. 1994 Sep 15;1214(2):148-60. doi: 10.1016/0005-2760(94)90039-6.
In the presence of taurodeoxycholate, cis-unsaturated fatty acids increase porcine pancreatic lipase activity 15-fold at pH 7.5. This effect is saturable with a low proportion of fatty acid to substrate. The overall angle of the fatty acid, the position of its double bond and the presence of a carboxyl group were critical factors in whether the fatty acid effectively increased lipase activity. When the substrate is emulsified by taurodeoxycholate, the pH optimum for lipase ranges from 6.2 to 7.0. In the presence of cis-unsaturated fatty acids, the overall activity of lipase increases, the pH optimum shifts, and the pH-activity curve becomes biphasic, with one optimum around pH 7.7, and the other around pH 8.8. Fluorescence studies indicate that fatty acids bind near aromatic residues in lipase, particularly tryptophan. Using the fluorescent fatty acid cis-parinaric acid, it was determined that multiple binding sites are present with Kd values of approx. 10(-6) M. Far-UV circular dichroism (CD) studies indicate that in addition to a high affinity fatty acid binding site with a Kd of approx. 10(-6) M, there is also a low affinity binding site with a Kd of approx. 10(-4) M. The far-UV CD data also show that cis-unsaturated fatty acids change the conformation of lipase. It is calculated that the percentage of alpha helix decreases, and the amount of beta sheet and beta turn structure increases. Because the three-dimensional crystal structure of lipase is known, a model is proposed to describe how cis-unsaturated fatty acids increase lipase activity.
在牛磺脱氧胆酸盐存在的情况下，顺式不饱和脂肪酸在pH 7.5时可使猪胰脂肪酶活性提高15倍。这种效应在脂肪酸与底物比例较低时即可饱和。脂肪酸的整体角度、双键位置以及羧基的存在是脂肪酸能否有效提高脂肪酶活性的关键因素。当底物被牛磺脱氧胆酸盐乳化时，脂肪酶的最适pH范围为6.2至7.0。在顺式不饱和脂肪酸存在的情况下，脂肪酶的整体活性增加，最适pH发生偏移，pH-活性曲线变为双相，一个最适pH约为7.7，另一个约为8.8。荧光研究表明，脂肪酸结合在脂肪酶的芳香族残基附近，尤其是色氨酸。使用荧光脂肪酸顺式十八碳四烯酸，确定存在多个结合位点，其解离常数（Kd）约为10⁻⁶ M。远紫外圆二色性（CD）研究表明，除了存在一个解离常数约为10⁻⁶ M的高亲和力脂肪酸结合位点外，还存在一个解离常数约为10⁻⁴ M的低亲和力结合位点。远紫外CD数据还表明，顺式不饱和脂肪酸会改变脂肪酶的构象。据计算，α螺旋的百分比降低，β折叠和β转角结构增加。由于脂肪酶的三维晶体结构已知，因此提出了一个模型来描述顺式不饱和脂肪酸如何提高脂肪酶活性。
