北京棒杆菌天冬氨酸激酶突变体A380H的酶学性质

Enzymatic Properties of Aspartate Kinase Mutant A380H from Corynebacterium pekinense

同源序列比对和蛋白质结构分析表明,A380为天冬氨酸激酶(aspartate kinase,AK)的绝对保守位点,对该位点进行定点突变、分离纯化和性质表征.结果表明:与野生型(WT)相比,突变体A380H的V_(max)提高4.28倍;突变体A380H的最适温度由28℃提高至35℃,最适pH值仍为7.5,半衰期由4.5h缩短至3.5h;底物抑制剂对WT和突变体均有抑制作用,苏氨酸和赖氨酸呈协同抑制作用,苏氨酸单独存在时对A380H具有激活或抑制减弱作用;Mg^(2+),Ni ^(2+)对WT有激活作用,1,5mmol/L的Cu^(2+)对A380H有激活作用;与WT相比,甲醇和异丙醇对A380H的抑制作用增强,正丁醇和乙腈对A380H的抑制作用减弱且表现出激活作用.

By homologous sequence and protein structure analysis,we found that A380 was an absolutely conserved site of aspartate kinase,and performed the site-directed mutagenesis,isolation,purification and characterization of the site.The results show that compared with the wild type（WT）,the V（max）of the mutant A380 His increased by 4.28 folds.The optimum temperature of the mutant A380 His increased from 28 ℃ to 35 ℃,the optimum pH is still 7.5,and the half-life is shortened from 4.5h to 3.5h.The substrate inhibitors have inhibitory effects on WT and mutants,threonine and lysine show synergistic inhibition effects.However,threonine alone has an activation or inhibitory effect on A380 H,Mg^2＋ and Ni^2＋ have an activation effect on WT,1,5mmol/L of Cu^2＋ has an activation effect on A380 H.Compared with WT,the inhibitory effect of methanol and isopropanol on A380 His enhanced.The inhibitory effect of n-butanol and acetonitrile on A380 His reduced and show activation.