Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene.
Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene.
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DOI：
10.1016/s0021-9258(19)62682-7
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发表时间：
1986-05
期刊：
影响因子：
--
通讯作者：
B. Hove-Jensen;K. Harlow;C. King;R. Switzer
中科院分区：
文献类型：
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作者：
B. Hove-Jensen;K. Harlow;C. King;R. Switzer
大肠杆菌磷酸核糖焦磷酸（P-Rib-PP）合成酶已从一株携带有编码P-Rib-PP合成酶的β-内酰胺酶基因的多拷贝质粒中纯化至接近均一性。对该酶的分析表明，它需要无机磷酸盐才能具有活性和稳定性。需要镁离子作为与底物ATP的复合物和作为游离阳离子。ADP强烈抑制P-Rib-PP合成酶活性。动力学分析表明ADP具有多个作用位点。此外，在ADP存在下，5-磷酸核糖产生明显的底物抑制。对E.大肠杆菌EclB基因的克隆及编码片段的确定。推导的P-Rib-PP合成酶的氨基酸序列包含314个氨基酸残基，计算的分子量为34，060。确定转录起始位点。该位点之前是高度保守的-10和-35共有序列（分别为pdT-dA-dG-dA-dA-dT和pdT-dT-dG-dA-dT-dG）。转录起始位点位于潜在翻译起始位点之前302个核苷酸。转录终止于UAA翻译终止密码子下游约35个核苷酸处，位于反向重复序列后的富Thy区域内，表明为rho非依赖性转录终止子。
Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability. Magnesium ions were required both as a complex with the substrate ATP and as a free cation. P-Rib-PP synthetase activity was inhibited strongly by ADP. Kinetic analysis indicated multiple sites of action of ADP. In addition apparent substrate inhibition was exerted by ribose 5-phosphate in the presence of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined. This site was preceded by well conserved -10 and -35 consensus sequences (pdT-dA-dG-dA-dA-dT and pdT-dT-dG-dA-dT-dG, respectively). The transcription initiation site preceded the potential translation initiation site by 302 nucleotides. Transcription terminated approximately 35 nucleotides downstream from the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator.