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首页> 外文期刊>Biochemistry >Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis
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Selective alteration of the rate-limiting step in cytosolic aldehyde dehydrogenase through random mutagenesis

机译：通过随机诱变选择性改变胞质醛脱氢酶的限速步骤

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摘要

Random mutagenesis followed by a filter-based screening assay has been used to identify a mutant of human class 1 aldehyde dehydrogenase (ALDH1) that was no longer inhibited by Mg2+ ions but was activated in their presence. Several mutants possessed double, triple, and quadruple amino acid substitutions with a total of seven different residues being altered, but each had a common T244S change. This point mutation proved to be responsible for the Mg2+ ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg2+ ions have been shown to increase the rate of deacylation. Consistent with the rate-limiting step for ALDH1 being changed from coenzyme dissociation to deacylation was finding that chloroacetaldehyde was oxidized more rapidly than acetaldehyde. Furthermore, Mg2+ ions only in the presence of NAD(H) increased the rate of hydrolysis of p-nitrophenyl acetate showing that the metal only affects the binary complex. Though the rate-limiting step for the T244S mutant was different from that of the native enzyme, the catalytic efficiency of the mutant was just 20% that of the native enzyme. The basis for the change in the rate-limiting step appears to be related to NAD(+) binding. Using the structure of a sheep class 1 ALDH, it was possible to deduce that the interaction between the side chain of T244 and its neighboring residues with the nicotinamide ring of NAD(+) were an essential determinant in the catalytic action of ALDH1.
机译：随机诱变，然后进行基于过滤器的筛选分析已用于鉴定人类1类乙醛脱氢酶（ALDH1）的突变体，该突变体不再受Mg2 +离子的抑制，但在其存在下被激活。几个突变体具有双，三和四个氨基酸取代，共有七个不同的残基被改变，但每个突变体都有一个共同的T244S变化。事实证明，这一点突变是导致Mg2 +离子活化的原因。重组表达ALDH1 T244S突变体，并用于机理研究。已显示Mg2 +离子会增加脱酰速率。与将ALDH1从辅酶解离变为脱酰作用的限速步骤一致，发现氯乙醛的氧化速度比乙醛快。此外，仅在NAD（H）存在下，Mg2 +离子会增加对硝基苯乙酸酯的水解速率，表明该金属仅影响二元络合物。尽管T244S突变体的限速步骤与天然酶不同，但该突变体的催化效率仅为天然酶的20％。限速步骤中更改的基础似乎与NAD（+）绑定有关。使用绵羊1类ALDH的结构，可以推断T244的侧链及其相邻残基与NAD（+）的烟酰胺环之间的相互作用是ALDH1催化作用的重要决定因素。

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