Sirawaraporn W, Edman J C, Santi D V
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Protein Expr Purif. 1991 Oct-Dec;2(5-6):313-6. doi: 10.1016/1046-5928(91)90088-z.
Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.
用甲氨蝶呤-琼脂糖亲和层析一步法将在大肠杆菌中表达的卡氏肺孢子虫二氢叶酸还原酶（DHFR）纯化至同质。纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上以单一的24 kDa蛋白迁移。纯化酶N端前26个氨基酸的序列与从DNA序列预测的序列一致。该酶表现出较宽的最适pH值，在pH 6至7范围内活性最高。该酶被盐激活，在50至150 mM KCl和50至200 mM NaCl时最大激活两倍。2.5 M的尿素也使酶活性增加两倍。对纯化酶的动力学分析表明，二氢叶酸和NADPH的Km值分别为1.8和1.4 μM，kcat为70 s-1。抑制研究证实，甲氧苄啶和乙胺嘧啶是卡氏肺孢子虫DHFR的弱抑制剂，对人DHFR几乎没有选择性。三甲曲沙和吡利曲辛是卡氏肺孢子虫酶更有效的抑制剂，但这些抑制剂也是人DHFR的有效抑制剂。
