亚甲基四氢叶酸还原酶的纯化及催化性质研究

Purification and Catalytic Properties of Methylenetetrahydrofolate Reductase

对亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)的体外酶促反应性质进行研究。通过盐析、DEAE-Sepharose fast flow柱层析纯化猪肝中的MTHFR,采用荧光法分析测定该酶纯化结果和酶促动力学性质。结果表明,MTHFR的纯化倍数可达20倍以上;测得最适酶促反应条件为pH 6.8,37℃;NADPH浓度1.2 mmol/L;以5,10-亚甲基四氢叶酸为底物测得米氏常数km=70.3μmol/L。研究发现,别构抑制剂S-腺苷甲硫氨酸(AdoMet)可使MTHFR的体外活性降到原活性的60%以下。本文初步探讨了MTHFR的催化性质,对MTHFR催化活性的主要影响因素进行研究,为酶促法合成5-甲基四氢叶酸的进一步研究奠定了基础。

To study the enzymatic reaction properties of methylenetetrahydrofolate reductase （MTHFR） in vitro, MTHFR was purified from porcine liver by salting out and DEAE-Sepharose fast flow column chromatography. The purification and kinetic characters of MTHFR were analyzed by flourescence method. The purification fold was more than 20. Property research showed that the optimum pH value and temperature of MTHFR were 6.8 and 37 ℃ respectively and the optimum NADPH concentration was 1.2 mmol/L. The krn values of MTHFR by using 5,10 - methylenetetrahydrofolate as the substrat was 70. 3 μmol/L. The results also indicate that allosteric inhibitor S-adenosylmethionine （AdoMet） can reduce the activity of MTHFR down to 60% compared with the original activity in vitro. This paper discussed the catalytic properties of MTHFR, as well as the main factors which influenced the activity of MTHFR. The preliminary study of the MTHFR mentioned above was the basis for the enzymatic synthesis of 5 - methylenetetrahydrofolate in the following researches.