Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change.
Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change.
复制标题
通过用丙氨酸替换 Asp222 来表征大肠杆菌天冬氨酸转氨酶中诱导的表观负协同性。
DOI：
10.1093/protein/7.3.413
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Kirsch,JF
中科院分区：
文献类型：
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作者：
Onuffer,JJ;Kirsch,JF
在大肠杆菌中，与天冬氨酸氨基转移酶(AATase)的吡哆醛5‘磷酸(PLP)辅助因子(PLP)的吡啶氮原子形成氢键盐桥的活性中心残基Asp222被丙氨酸(D222A)取代。D222a突变体与氨基酸底物呈现非双曲线饱和行为，在稳态动力学分析中表现为明显的负相互作用。D222A的单周转过程曲线可用两个指数之和很好地描述，与野生型酶的单相动力学相反。含有D222A突变的活性/非活性异二聚体保留了这一双相动力学反应，证明所观察到的同功性不是诱导变构的结果。这种异常行为可以用滞后动力学模型来解释，该模型涉及两种缓慢相互转化的酶，其中只有一种是催化活性的。这两种形式之间的缓慢功能转换的半衰期为∼10分钟。将突变体与二羧基抑制剂马来酸盐预先孵育，使酶的平衡种群向催化活性形式转移，表明缓慢的转变与已知的当该抑制剂与野生型酶结合时发生的结构域关闭有关。Asp222在转氨化的化学步骤中的重要性从D222A突变体中∼的催化活性降低了105倍和大的初级Cα-H动力学同位素效应(野生型为6.7vs2.2)中得到证实。通过与辅因子类似物N-甲基吡哆醛-5-磷酸(N-α)的重组，D222A突变体的转氨酶活性提高了4~20倍，C-MPLP-质子提取步骤的速率决定较少，初级动力学同位素效应从6.7%下降到2.3%。这些结果表明，PLP的质子化吡啶氮与ASP222的带负电荷的羧酸盐之间的保守相互作用不仅对于高效的Cα-质子提取是重要的，而且对于伴随转氨化过程的构象转变也是重要的
The strictly conserved active site residue, Asp222, which forms a hydrogen-bonded salt bridge with the pyridine nitrogen atom of the pyridoxal 5′ phosphate (PLP) co-factor of aspartate aminotransferase (AATase), was replaced with alanine (D222A) in theEscherichia colienzyme. The D222A mutant exhibits non–hyberbolic saturation behavior with amino acid substrates which appear as apparent negative eooperativity in steady–state kinetic analyses. Single turnover progress curves for D222A are well described by the sum of two exponentials, contrasting with the monophasic kinetics of the wild-type enzyme. An active/inactive heterodimer containing the D222A mutation retains this biphasic kinetic response, proving that the observed eooperativity is not the result of induced allostery. The anomalous behavior is explained by a hysteretic kinetic model involving two slowly interconverting enzyme forms, only one of which is catalytically competent. The slow functional transition between the two forms has a half–life of ∼ 10 mins. Preincubation of the mutant with the dicarboxylk inhibitor maleate shifts the equilibrium population of the enzyme towards the catalytically active form, suggesting that the slow transition is related to the domain closure known to occur upon association of this inhibitor with the wild-type enzyme. The importance of Asp222 in the chemical steps of transamination is confirmed by the ∼l05fold decrease in catalytic competence in the D222A mutant, and by the large primary Cα–deuterium kinetic isotope effect (6.7 versus 2.2 for the wild–type). The transamination activity of the D222A mutant is enhanced 4– to 20–fold by reconstltution with the co-factor analogN–methylpyridoxal–5–phosphate (N–MePLP), and the Cα–proton abstraction step is less rate determining, as evidenced by the decrease in the primary kinetic isotope effect from 6.7 to 2.3. These results suggest that the conserved interaction between the protonated pyridine nitrogen of PLP and the negatively charged carboxylate of Asp222 is important not only for efficient Cα–proton abstraction, but also for conformational transitions concomitant with the transamination process