Acidic Residues Important for Substrate Binding and Cofactor Reactivity in Eukaryotic Ornithine Decarboxylase Identified by Alanine Scanning Mutagenesis (*)
Acidic Residues Important for Substrate Binding and Cofactor Reactivity in Eukaryotic Ornithine Decarboxylase Identified by Alanine Scanning Mutagenesis (*)
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DOI：
10.1074/jbc.270.20.11797
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发表时间：
1995-05
期刊：
影响因子：
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通讯作者：
A. Osterman;L. Kinch;N. Grishin;M. Phillips
中科院分区：
文献类型：
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作者：
A. Osterman;L. Kinch;N. Grishin;M. Phillips
来自布氏锥虫、小鼠和杜氏利什曼原虫的鸟氨酸脱羧酶对三种碱性氨基酸（鸟氨酸、赖氨酸和精氨酸）具有严格的特异性。为了鉴定参与该底物特异性和/或反应化学的残基，将T中的6个保守酸性残基（Asp-88、Glu-94、Asp-233、Glu-274、Asp-361和Asp-364）突变为丙氨酸。布氏杆菌酶每个突变都会导致酶效率的大幅下降。最值得注意的是，突变的Asp-361增加了2000倍的K为鸟氨酸，与K的影响不大，这表明该残基是一个重要的底物结合决定簇。突变唯一严格保守的酸性残基Glu-274，使k降低50倍;然而，将N-甲基吡哆醛-5 ′-磷酸替换为吡哆醛-5 ′-磷酸作为反应中的辅因子，使E274 A的k恢复到野生型水平。这些数据证明Glu-274与辅因子的质子化吡啶氮相互作用以增强环的吸电子能力，类似于天冬氨酸转氨酶中的Asp-222（Onuffer，J. J.，和Kirsch，J. F.（1994）Protein Eng.7，413-424）。真核生物鸟氨酸脱羧酶是一种同源二聚体，具有两个共有的活性位点。残基88、94、233和274来自与Lys-69相同的亚基，而残基361和364是Cys-360亚基的一部分。
Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Kfor ornithine by 2000-fold, with little effect on k, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases k 50-fold; however, substitution of N-methylpyridoxal-5′-phosphate for pyridoxal-5′-phosphate as the cofactor in the reaction restores the k of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F.(1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.