INVOLVEMENT OF GLUTAMATE-268 IN THE ACTIVE-SITE OF HUMAN LIVER MITOCHONDRIAL (CLASS-2) ALDEHYDE DEHYDROGENASE AS PROBED BY SITE-DIRECTED MUTAGENESIS
INVOLVEMENT OF GLUTAMATE-268 IN THE ACTIVE-SITE OF HUMAN LIVER MITOCHONDRIAL (CLASS-2) ALDEHYDE DEHYDROGENASE AS PROBED BY SITE-DIRECTED MUTAGENESIS
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DOI：
10.1021/bi00001a028
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发表时间：
1995-01-10
期刊：
影响因子：
2.9
通讯作者：
WEINER, H
中科院分区：
文献类型：
--
作者：
WANG, XP;WEINER, H
基于化学修饰研究，假定谷氨酸268是肝醛脱氢酶活性位点的组分[Abriola，D. P.，菲尔兹河，Mackerell，A. D.，小的，和Pietruszko，R.（1987）Biochemistry 26，5679-5684]。为了研究其作用，将人肝线粒体（2类）醛脱氢酶中的残基突变为天冬氨酸、谷氨酰胺或赖氨酸，并在大肠杆菌中表达该酶。突变不影响NAD或丙醛的K-m值，但与重组表达的天然酶相比，突变体酶显著影响酶的催化活性;突变体酶的比活性小于重组表达的天然醛脱氢酶的0.4%。突变还导致在反应的稳态阶段之前发生长的滞后阶段。突变酶的活性不能通过添加一般碱如乙酸钠、甲酸钠或咪唑来恢复。对于E268 Q突变体和天然酶，NADH的Kd基本上相同。三种突变形式的酶具有小于0.8%的重组表达的天然酶的酯水解活性。预稳态分析表明，在脱氢酶反应中没有NADH形成的爆发或在酯酶反应中没有对硝基苯酚形成的爆发。这可以解释为暗示谷氨酸268可以作为必需半胱氨酸残基（302）的初始活化所必需的一般碱起作用，而不是仅参与脱酰或氢化物转移步骤。或者，谷氨酸268可以作为激活亲核残基所需的电荷中继三联体的组分。此外，看来酯酶和脱氢酶需要相同的活性位点组分，因为当谷氨酸268被改变为另一个残基时，脱氢酶活性和酯酶活性基本上被消除。
On the basis of chemical modification studies, it was postulated that glutamate 268 was a component of the active site of liver aldehyde dehydrogenase [Abriola, D.P., Fields, R., MacKerell, A.D., Jr., and Pietruszko, R. (1987) Biochemistry 26, 5679-5684]. To study its role, the residue in human liver mitochondrial (class 2) aldehyde dehydrogenase was mutated to an aspartate, a glutamine, or a lysine, and the enzyme was expressed in Escherichia coli. The mutations did not affect the K-m values for NAD or propionaldehyde, but grossly affected the catalytic activity of the enzymes when compared to recombinantly expressed native enzyme; the mutant enzymes had less that 0.4% of the specific activity of the recombinantly expressed native aldehyde dehydrogenase. The mutations also caused a long lag phase to occur prior to the steady state phase of the reaction. The activity of the mutant enzymes could not be restored by the addition of general bases such as sodium acetate, sodium formate, or imidazole. The K-d for NADH was essentially identical for the E268Q mutant and native enzyme. The three mutant forms of the enzyme possessed less than 0.8% of the esterolytic activity of the recombinantly expressed native enzyme. Pre-steady state analysis showed that there was no burst of NADH formation in the dehydrogenase reaction or of p-nitrophenol formation in the esterase reaction. This can be interpreted as implying that glutamate 268 may function as a general base necessary for the initial activation of the essential cysteine residue (302), rather than being involved in only the deacylation or hydride transfer step. Alternatively, glutamate 268 could function as a component of a charge relay triad necessary to activate the nucleophilic residue. Furthermore, it appears that esterase and dehydrogenase require the same active site components, for both the dehydrogenase activity and esterase activity were essentially abolished when glutamate 268 was changed to another residue.