Farrés J, Wang X, Takahashi K, Cunningham S J, Wang T T, Weiner H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1994 May 13;269(19):13854-60.
Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.
许多东方人拥有一种肝脏线粒体醛脱氢酶，其487位的谷氨酸被赖氨酸取代，且他们的线粒体醛脱氢酶活性水平极低。为研究这种醛脱氢酶活性缺失的原因，我们将大鼠和人类肝脏线粒体醛脱氢酶的487位残基突变为赖氨酸，并在大肠杆菌中表达突变型和天然型酶形式。大鼠和人类重组醛脱氢酶均表现出与从肝脏线粒体分离出的酶相同的分子和动力学特性。与谷氨酸酶相比，发现E487K突变体具有活性但动力学特性发生了改变。在pH 7.4时，NAD⁺的Km增加了150多倍，而相对于重组天然酶，kcat降低了2至10倍。详细的稳态动力学分析表明，NAD⁺与突变酶的结合受损，并且可以计算出这导致活性位点半胱氨酸残基的亲核性降低。大鼠E487K突变体的限速步骤也与重组大鼠肝脏醛脱氢酶不同，因为突变酶未发现NADH形成的稳态前爆发。大鼠天然酶和E487K突变体氧化氯乙醛的速度均是乙醛的两倍，这表明限速步骤不是氢化物转移或辅酶解离，而是取决于亲核攻击。每种酶形式在添加Mg²⁺离子后均表现出2倍的激活。用谷氨酰胺取代谷氨酸对酶的性质没有明显影响。487位谷氨酸可能直接与NAD⁺的正烟酰胺环相互作用，因为赖氨酸酶中NADH的Ki与谷氨酸形式相同。由于E487K变体的NAD⁺结合特性和kcat发生了改变，推测拥有这种形式的人将没有功能性的线粒体醛脱氢酶。
