Fusek M, Lin X L, Tang J
Laboratory of Protein Studies, Oklahoma Medical Research Foundation, Oklahoma City.
J Biol Chem. 1990 Jan 25;265(3):1496-501.
The specificity of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius, was studied using oxidized insulin B chain as substrate followed by peptide isolation and identification. The following bonds were hydrolyzed: Leu-Val, Leu-Tyr, Phe-Phe, Phe-Tyr, and Tyr-Thr. Thus, the specificity of thermopsin is similar to that of pepsin, that is, it prefers large hydrophobic residues at both sides of the scissile bond. We confirmed this by the use of a synthetic substrate, Lys-Pro-Ala-Glu-Phe-p-nitro-phenylalanyl-Ala-Leu, which was cleaved by thermopsin between Phe and p-nitro-phenylalanyl. Using this substrate, enzyme inhibition and kinetic properties of thermopsin have been studied. Thermopsin optimally hydrolyzes this substrate at 75 degrees C and pH 2 with Km and kcat values under these conditions of 5.3 x 10(-5) M and 14.3 s-1, respectively. Pepstatin competitively inhibits thermopsin with a Ki of 2 x 10(-7) M. Other known aspartic protease inhibitors, diazoacetylnorleucine ethyl ester and 1,2-epoxy-3-(p-nitrophenoxy)propane inhibited thermopsin only slowly and with nonspecific reactions. Although thermopsin contains a single cysteine, iodoacetic acid and p-chloromercuric benzoate had no effect on activity. Mercuric chloride inhibited the enzyme, and the inhibition was reversible by mercaptoethanol. However, the enzyme was not labeled by [14C]iodoacetic acid either before or after sodium dodecyl sulfate denaturation. Thus, the thiol group is likely blocked, and the inhibition effect of mercuric ion is unrelated to the thiol group. These observations suggest that thermopsin has a different active site than the aspartic protease family but may have a similar transition state structure. The temperature dependence of Km and kcat was studied for thermopsin hydrolysis of the synthetic substrate between 26-78 degrees C. Both parameters increased with temperature, and the rise of kcat value was particularly sharp above 65 degrees C. Hydrolysis activity measured at high substrate concentration has a maximum at 76 degrees C, which is near the physiological temperature for the optimal growth of this organism. Thus, thermopsin appears to function best at high temperature and high substrate concentration. It may be utilized by the organism to response to the presence of high substrate concentration in the medium. Thermopsin is also competitively inhibited by urea, acetamide, and phenylalaninamide with Ki values of 0.5, 0.4, and 0.01 M, respectively.
以氧化胰岛素B链为底物，通过肽段分离和鉴定，研究了嗜酸热硫化叶菌（Sulfolobus acidocaldarius）中一种热稳定酸性蛋白酶——嗜热蛋白酶的特异性。以下肽键被水解：亮氨酸-缬氨酸、亮氨酸-酪氨酸、苯丙氨酸-苯丙氨酸、苯丙氨酸-酪氨酸和酪氨酸-苏氨酸。因此，嗜热蛋白酶的特异性与胃蛋白酶相似，即它倾向于在裂解键两侧有大的疏水残基。我们通过使用合成底物赖氨酸-脯氨酸-丙氨酸-谷氨酸-苯丙氨酸-对硝基苯丙氨酰-丙氨酸-亮氨酸证实了这一点，该底物在嗜热蛋白酶作用下在苯丙氨酸和对硝基苯丙氨酰之间被裂解。利用该底物，研究了嗜热蛋白酶的酶抑制作用和动力学性质。嗜热蛋白酶在75℃和pH 2条件下对该底物的水解作用最佳，在此条件下Km和kcat值分别为5.3×10⁻⁵M和14.3 s⁻¹。胃酶抑素竞争性抑制嗜热蛋白酶，Ki为2×10⁻⁷M。其他已知的天冬氨酸蛋白酶抑制剂，重氮乙酰正亮氨酸乙酯和1,2-环氧-3-(对硝基苯氧基)丙烷对嗜热蛋白酶的抑制作用缓慢且伴有非特异性反应。虽然嗜热蛋白酶含有一个半胱氨酸，但碘乙酸和对氯汞苯甲酸对其活性没有影响。氯化汞抑制该酶，而巯基乙醇可使抑制作用逆转。然而，在十二烷基硫酸钠变性前后，该酶均未被[¹⁴C]碘乙酸标记。因此，巯基可能被封闭，汞离子的抑制作用与巯基无关。这些观察结果表明，嗜热蛋白酶与天冬氨酸蛋白酶家族具有不同的活性位点，但可能具有相似的过渡态结构。研究了26 - 78℃下嗜热蛋白酶对合成底物水解时Km和kcat的温度依赖性。两个参数均随温度升高而增加，kcat值在65℃以上的升高尤为明显。在高底物浓度下测得的水解活性在76℃时达到最大值，这接近该生物体最佳生长时的生理温度。因此，嗜热蛋白酶似乎在高温和高底物浓度下功能最佳。它可能被该生物体用于响应培养基中高底物浓度的存在。嗜热蛋白酶也受到尿素、乙酰胺和苯丙氨酰胺的竞争性抑制，Ki值分别为0.5、0.4和0.01 M。
