The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid.
The effect of glyoxalase I on the metabolism of 4,5-dioxovaleric acid.
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乙二醛酶 I 对 4,5-二氧戊酸代谢的影响。
DOI：
10.1016/0304-4165(85)90036-4
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Burnham,BF
中科院分区：
文献类型：
--
作者：
Stafforini,DM;Kushner,JP;Burnham,BF
哺乳动物细胞中5-氨基乙酰丙酸的生物合成是由氨基乙酰丙酸合成酶通过甘氨酸和琥珀酰辅酶a的缩合反应催化的。哺乳动物细胞中的另一种途径可能涉及氨基乙酰丙酸的生物合成，其中丙氨酸是氨基供体，4,5-二氧戊酸是受体。这种转氨化反应，或一个非常类似的反应，被植物用于氨基乙酰丙酸的生物合成，最终转化为叶绿素。用纯化的乙二醛酶I和粗组织匀浆测试了乙二醛酶I对二氧戊酸转化为其他产品的影响。乙二醛酶I是一种金属酶，谷胱甘肽是一种共底物。纯化的乙草醛酶I减少了在二氧戊酸、l -丙氨酸、谷胱甘肽和纯化的l -丙氨酸：4,5-二氧戊酸转氨酶（二氧戊酸转氨酶）存在下形成的氨基乙酰丙酸的数量。二氧戊酸向氨基乙酰丙酸的转化被添加谷胱甘肽所抑制，当透析的牛肝脏匀浆同时作为乙草醛酶I和二氧戊酸转氨酶的来源时。从牛肝脏匀浆中去除金属可使乙二醛酶I活性降低85%。在MgSO4预孵育后，这些“无金属”匀浆仍然影响二氧戊酸向氨基乙酰丙酸的转化。通过偶联酶反应将二氧戊酸转化为尿卟啉，利用荧光酶法定量测定二氧戊酸，研究了乙草醛酶I对二氧戊酸代谢的影响。肝脏和大麦的匀浆都减少了偶联试验检测到的二氧戊酸的量，但这种影响可以通过透析匀浆来防止。在透析匀浆中加入谷胱甘肽可显著降低二氧戊酸产生的尿卟啉的量。在偶联实验中，添加谷胱甘肽的无金属匀浆降低了二氧戊酸向尿卟啉的转化，但用mgso4预孵育大大增强了这一作用。这些研究指出了用全细胞匀浆评价二氧戊酸作为血红素前体的困难。
Biosynthesis of 5-aminolevulinic acid in mammalian cells is catalyzed by aminolevulinic acid synthase in a condensation reaction utilizing glycine and succinyl · coenzyme A. An alternate pathway in mammalian cells may involve the biosynthesis of aminolevulinic acid via a transamination reaction in whichL-alanine is the amino donor and 4,5-dioxovaleric acid is the acceptor. This transamination reaction, or one very similar, is employed by plants for the biosynthesis of aminolevulinic acid which is ultimately converted to chlorophyll. The effect of glyoxalase I on the diversion of dioxovaleric acid to other products was tested using both purified glyoxalase I and crude tissue homogenates. Glyoxalase I is a metalloenzyme and glutathione is a co-substrate. Purified glyoxalase I reduced the amount of aminolevulinic acid formed in the presence of dioxovaleric acid,L-alanine, glutathione, and purifiedL-alanine: 4,5-dioxovaleric acid aminotransferase (dioxovalerate transaminase). The conversion of dioxovaleric acid to aminolevulinic acid was inhibited by the addition of glutathione when a dialyzed bovine liver homogenate served as the source of both glyoxalase I and dioxovalerate transaminase. Removal of metals from bovine liver homogenates produced an 85% decrease in glyoxalase I activity. These ‘metal-free’ homogenates still affected the conversion of dioxovaleric acid to aminolevulinic acid after preincubation with MgSO4. The effect of glyoxalase I on the metabolism of dioxovaleric acid was also studied using a fluorometric enzyme assay for the quantification of dioxovaleric acid via a coupled enzyme reaction converting it to uroporphyrin. Homogenates of both liver and barley diminished the amount of dioxovaleric acid detected by the coupled assay, but this effect could be prevented by dialysis of the homogenates. Addition of glutathione to dialyzed homogenates markedly reduced the amount of uroporphyrin generated from dioxovaleric acid. Metal-free homogenates supplemented with glutathione reduced the conversion of dioxovaleric acid to uroporphyrin in the coupled assay, but preincubation with MgSO4greatly augmented this effect. These studies point out the difficulty in evaluating dioxovaleric acid as a heme precursor using whole cell homogenates.