苹果酸酶结合域定点突变对烟酰胺腺嘌呤二核苷酸类似物催化性能的影响

doi:10.11944/j.issn.1000-0518.2016.04.150268

摘要/Abstract

摘要：

通过对苹果酸酶(ME)辅酶结合域L310、Q401、L404饱和位点突变库与辅酶烟酰胺腺嘌呤二核苷酸(NAD⁺)类似物库的高通量筛选,研究了苹果酸酶结合域位点对NAD⁺及其类似物(B1~B7)催化活性的影响。结果表明,突变后酶ME-Q401H/L404T对类似物B4的k_cat/K_m是野生型酶的50倍;突变后酶ME-L310M/Q401N对类似物B4的k_cat/K_m是野生型酶的16倍,对类似物B3的k_cat/K_m是野生型酶的5倍,因此通过对结合域定点突变,NAD⁺类似物的催化活性得到提高。

关键词: 苹果酸酶, 定点突变, 辅酶活性

Abstract:

The cofactor-binding domains of malic enzyme(ME) L310, Q401 and L404 were found to have a steric effect on binding of nicotinamide adenine dinucleotide(NAD⁺). The site-directed mutagenesis of these three sites shows that the cofactor activities of NAD⁺ analogs(B1~B7) change in some level, indicating that these sites have a critical role in binding the cofactors. A high-throughput screen between malic enzyme mutants and NAD⁺ analogs affords 2 different mutants capable of taking NAD⁺ analogs as the cofactor. ME-Q401H/L404T has a 50-fold higher k_cat/K_m than the that of wild-type ME for the analog B4. For ME-L310M/Q401N, the k_cat/K_m are 16-fold and 5-fold higher than those of wild-type ME for the cofactors B4 and B3. The coenzyme activity can be increased through site-directed mutagenesis of cofactor-binding domains.

Key words: malic enzyme, site-directed mutagenesis, coenzyme activity

侯淑华, 纪德彬, 刘武军, 王磊. 苹果酸酶结合域定点突变对烟酰胺腺嘌呤二核苷酸类似物催化性能的影响[J]. 应用化学, 2016, 33(4): 436-441.

HOU Shuhua, JI Debin, LIU Wujun, WANG Lei. Effect of Site-Directed Mutagenesis in Coenzyme-Binding Domain of Malic Enzyme on Coenzyme Activities for Nicotinamide Adenine Dinucleotide Analogs[J]. Chinese Journal of Applied Chemistry, 2016, 33(4): 436-441.

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