Molecular enzymology of mammalian Δ1-pyrroline-5-carboxylate synthase -: Alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition
Molecular enzymology of mammalian Δ1-pyrroline-5-carboxylate synthase -: Alternative splice donor utilization generates isoforms with different sensitivity to ornithine inhibition
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DOI：
10.1074/jbc.274.10.6754
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发表时间：
1999-03-05
影响因子：
4.8
通讯作者：
Valle, D
中科院分区：
文献类型：
--
作者：
Hu, CA;Lin, WW;Valle, D
δ（1）-吡咯啉-5-羧酸合酶（P5 CS; EC未指定）是一种线粒体内膜ATP和NADPH依赖性双功能酶，催化谷氨酸还原为δ（1）-吡咯啉-5-羧酸，这是脯氨酸和鸟氨酸从头生物合成的关键步骤。我们利用已发表的植物P5 CS序列搜索表达序列标签数据库，克隆了两个在开放阅读框中长度相差6个碱基对（bp）的全长人P5 CS cDNA。短cDNA具有2379-bp的开放阅读框，编码793个残基的蛋白质;通过“外显子滑动”（一种选择性剪接形式）产生的长cDNA在短形式的bp +711之后含有额外的6-bp插入物，导致在预测为P5 CS的γ-谷氨酰激酶活性位点的区域中包含两个额外的氨基酸，除肠外，在所有检查的组织中长型占主导地位。我们还分离了相应的长和短鼠P5 CS转录本。为了确认推定的P5 CS cDNA的身份，我们在γ-谷氨酰激酶和γ-谷氨酰磷酸还原酶缺陷的酿酒酵母菌株中表达了人类形式，并表明它们赋予脯氨酸原养型。此外，我们发现小鼠推定的P5 CS cDNA的表达赋予脯氨酸原养型P5 CS缺陷的中国仓鼠卵巢细胞（CHO-K1）。我们利用稳定的CHO-K1细胞转化体来比较长和短鼠P5 CS同种型的生物化学特征。我们发现两者都赋予P5 CS活性，并且短同种型被L-鸟氨酸抑制，Ki类似于0.25 mM。因此，长同种型中的两个氨基酸插入物消除了L-鸟氨酸对P5 CS活性的反馈抑制。
Delta(1)-Pyrroline-5-carboxylate synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP- and NADPH-dependent, bifunctional enzyme, catalyzes the reduction of glutamate to Delta(1)-pyrroline-5-carboxylate, a critical step in the de novo biosynthesis of proline and ornithine. We utilized published plant P5CS sequence to search the expressed sequence tag data base and cloned two full-length human P5CS cDNAs differing in length by 6 base pairs (bp) in the open reading frame. The short cDNA has a 2379-bp open reading frame encoding a protein of 793 residues; the long cDNA, generated by "exon sliding," a form of alternative splicing, contains an additional 6-bp insert following bp +711 of the short form resulting in inclusion of two additional amino acids in the region predicted to be the gamma-glutamyl kinase active site of P5CS, The long form predominates in all tissues examined except gut. We also isolated the corresponding long and short murine P5CS transcripts. To confirm the identity of the putative P5CS cDNAs, we expressed both human forms in gamma-glutamyl kinase- and gamma-glutamyl phosphate reductase-deficient strains of Saccharomyces cerevisiae and showed that they conferred the proline prototrophy. Additionally, we found expression of the murine putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell transformants to compare the biochemical characteristics of the long and short murine P5CS isoforms, We found that both confer P5CS activity and that the short isoform is inhibited by L-ornithine with a K-i of similar to 0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two amino acid insert in the long isoform abolishes feedback inhibition of P5CS activity by L-ornithine.