Chen Z, Koelsch G, Han H P, Wang X J, Lin X L, Hartsuck J A, Tang J
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City.
J Biol Chem. 1991 Jun 25;266(18):11718-25.
A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion.
一个包含完整根霉胃蛋白酶结构和假定前体区域的cDNA克隆被置于三种不同的大肠杆菌表达载体中，用于合成根霉胃蛋白酶原（Rpg）。在大肠杆菌胞质溶胶中作为包涵体表达的重组Rpg（cRpg和tRpg）没有活性。在6 M尿素中溶解并通过快速稀释复性后，这两种Rpg都被纯化至同质。第三种酶原pRpg通过omp前导序列分泌到大肠杆菌的周质空间，具有完全活性且也被纯化。pRpg的表达水平（超过40 mg/升培养物）高于cRpg（约1.5 mg/升培养物）。对这些酶原的氨基末端序列分析表明，cRpg和pRpg分别含有40和51个前导序列残基。在T7启动子控制下表达的tRpg在500 mg/升培养物中合成，并在50 mg/升培养物中纯化。除了51个前体区域残基外，这种酶原还含有从表达载体构建中继承的16个残基。所有这些Rpg在pH小于5的溶液中都会自发转化为根霉胃蛋白酶。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳监测下，每次转化都伴随着分子量的变化。对于cRpg和pRpg酶原，在2至3的pH范围内至少观察到一种转化中间体。对于pRpg和tRpg，动力学数据表明，在pH 2时，Rpg向根霉胃蛋白酶的转化是通过一级单分子反应完成的。在15℃下该pH时，pRpg和tRpg的一级动力学常数分别为1.1和2.4 min-1。随着pH升高到pH 2以上，活化速率降低。在pH大于3.0时，根霉胃蛋白酶催化的二级活化也会发生。因此，重组Rpg与胃蛋白酶原一样，通过两种裂解机制之一被激活。这些结果也支持了Rpg在华根霉中作为酶原合成的假说。宿主真菌中的Rpg可能在分泌颗粒中被pH小于5的酸性环境激活，在分泌前变成根霉胃蛋白酶。
