Berman J, Green M, Sugg E, Anderegg R, Millington D S, Norwood D L, McGeehan J, Wiseman J
Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1992 Jan 25;267(3):1434-7.
A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.
本文描述了一种快速优化蛋白酶底物催化常数（kcat）与米氏常数（Km）比值（kcat/Km）的策略。通过用氨基酸混合物合成来改变给定肽底物序列的选定位置。将所得肽混合物与目标酶一起孵育，并通过高压液相色谱进行分析，可直接测定kcat/Km增强的类似物。使用高效液相色谱/连续流快原子轰击质谱法来确定色谱图中每个峰的结构。作为“底物图谱”实用性和效率的一个例子，我们描述了在P'1和P'2位置对胶原酶底物二硝基苯基 - 脯氨酸 - 亮氨酸 - 甘氨酸 - 亮氨酸 - 色氨酸 - 丙氨酸 - D - 精氨酸 - 氨基（其中Dnp是二硝基苯基）的优化。制备了六种不同的混合物用于评估，代表了128种不同合成底物的合成。“底物图谱”已得到二硝基苯基 - 脯氨酸 - 亮氨酸 - 甘氨酸 - 半胱氨酸（甲基） - 组氨酸 - 丙氨酸 - D - 精氨酸 - 氨基，该底物的kcat/Km比二硝基苯基 - 脯氨酸 - 亮氨酸 - 甘氨酸 - 亮氨酸 - 色氨酸 - 丙氨酸 - D - 精氨酸 - 氨基高10倍。
