PURIFICATION AND CHARACTERIZATION OF THE PURE, PURK, AND PURC GENE-PRODUCTS - IDENTIFICATION OF A PREVIOUSLY UNRECOGNIZED ENERGY REQUIREMENT IN THE PURINE BIOSYNTHETIC-PATHWAY
PURIFICATION AND CHARACTERIZATION OF THE PURE, PURK, AND PURC GENE-PRODUCTS - IDENTIFICATION OF A PREVIOUSLY UNRECOGNIZED ENERGY REQUIREMENT IN THE PURINE BIOSYNTHETIC-PATHWAY
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DOI：
10.1021/bi00136a016
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发表时间：
1992-06-02
期刊：
影响因子：
2.9
通讯作者：
SMITH, JM
中科院分区：
文献类型：
--
作者：
MEYER, E;LEONARD, NJ;SMITH, JM
氨基咪唑核苷酸羧化酶是嘌呤生物合成途径中的第六步，催化氨基咪唑核苷酸（AIR）转化为羧氨基咪唑核苷酸（CAIR）。被认为负责这种活性的purE和purK基因（分别为PurE和PurK）的基因产物已被过表达，蛋白质被纯化至均一。在纯化过程中，PurE在第一次硫酸铵分馏中与PurK分离。使用各种方法在各种条件下无法获得两种基因产物相关的证据。为了便于CAIR生产的测定，还将purC基因产物5-氨基咪唑-4-N-琥珀酰甲酰胺核糖核苷酸（SAICAR）合成酶过表达并纯化至均一。研究了PurE、PurK和PurE.PurK的活性。PurE单独催化AIR转化为CAIR的速度比非酶催化的速度快100万倍。在PurE依赖性反应中，HCO 3-的K（m）为110 mM！PurK具有依赖于AIR存在的ATP酶活性。没有碳酸氢盐依赖于这个反应可以证明（< 100 μ M），和空气是不羧化的ATP水解过程中。将PurE和PurK的1：1混合物在低浓度碳酸氢盐（< 100 μ M）下孵育，显示产生CAIR，但需要ATP化学计量转化为ADP和P（i）。没有依赖于HCO 3-的浓度可以证明。嘌呤生物合成途径中的新的能量需求已经建立。
Aminoimidazole ribonucleotide carboxylase, the sixth step in the purine biosynthetic pathway, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR). The gene products of the purE and purK genes (PurE and PurK, respectively) thought to be responsible for this activity have been overexpressed and the proteins purified to homogeneity. PurE separates from PurK in the first ammonium sulfate fractionation during the purification. No evidence for association of the two gene products under a variety of conditions using a variety of methods could be obtained. To facilitate the assay for CAIR production, the purC gene product, 5-aminoimidazole-4-N-succinylcarboxamide ribonucleotide (SAICAR) synthetase has also been overexpressed and purified to homogeneity. The activities of PurE, PurK, and PurE.PurK have been investigated. PurE alone is capable of catalyzing the conversion of AIR to CAIR 1 million times faster than the nonenzymatic rate. The K(m) for HCO3- in the PurE-dependent reaction is 110 mM! PurK possesses an ATPase activity that is dependent on the presence of AIR. No bicarbonate dependence on this reaction could be demonstrated (< 100-mu-M), and AIR is not carboxylated during the hydrolysis of ATP. Incubation of a 1:1 mixture of PurE and PurK at low concentrations of bicarbonate (< 100-mu-M) revealed that CAIR is produced but requires the stoichiometric conversion of ATP to ADP and P(i). No dependence on the concentration of HCO3- could be demonstrated. A new energy requirement in the purine biosynthetic pathway has been established.