Altering the reaction specificity of eukaryotic ornithine decarboxylase
Altering the reaction specificity of eukaryotic ornithine decarboxylase
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DOI：
10.1021/bi001209s
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发表时间：
2000-09-19
期刊：
影响因子：
2.9
通讯作者：
Phillips, MA
中科院分区：
文献类型：
--
作者：
Jackson, LK;Brooks, HB;Phillips, MA
鸟氨酸脱羧酶（ODC）催化多胺生物合成的第一个关键步骤，并且已被确定为治疗由布氏锥虫引起的非洲昏睡病的药物靶标。ODC是吡哆醛5 '-磷酸（PLP）依赖性酶，是一种专性同源二聚体。具有产物腐胺的布氏野生型酶揭示了在配体结合时发生的两种结构变化：Lys-69被腐胺取代并与Glu-94和Asp-88形成新的相互作用，Cys-360的侧链旋转到活性位点中，在亚胺键的3.4埃内。Cys-360突变为Ala或Ser分别使脱羧反应的k（cat）降低50倍和1000倍。然而，产物的HPLC分析表明，突变酶几乎只催化脱羧依赖性转氨反应，形成吡哆胺5-磷酸（PMP）和γ-氨基丁醛，而不是PLP和腐胺。当脱羧的底物中间体在PLP的C4'而不是在底物的C-α处质子化时，出现该副反应。对于由野生型酶催化的反应，这种副反应很少发生（
Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer, X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 Angstrom of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C-alpha of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (