Messenger L J, Zalkin H
J Biol Chem. 1979 May 10;254(9):3382-92.
Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol.
谷氨酰胺5-磷酸核糖胺：焦磷酸磷酸核糖转移酶（酰胺磷酸核糖转移酶）已从大肠杆菌中纯化至同质。通过沉降平衡离心法测得天然酶的分子量为194,000，通过凝胶过滤法测得为224,000。在十二烷基硫酸钠中进行凝胶电泳估计亚基分子量为57,000。交联实验得到分子量为57,000、117,000和177,000的物种。表明天然酶为相同亚基的三聚体或四聚体。高活性的大肠杆菌酰胺磷酸核糖转移酶不含大量非血红素铁。添加铁盐和硫化物不会增强酶活性。酰胺磷酸核糖转移酶表现出依赖氨和谷氨酰胺的活性。在没有其他底物的情况下检测到谷氨酰胺酶活性。依赖谷氨酰胺和氨的活性均受到嘌呤5'-核糖核苷酸的终产物抑制。AMP和GMP共同作用产生协同抑制。AMP和GMP表现出正协同性。此外，GMP促进了5-磷酸核糖-1-焦磷酸饱和的协同性。氨抑制谷氨酰胺的利用，这表明在5-磷酸核糖-1-焦磷酸胺化之前，谷氨酰胺的酰胺基被转移到氨位点。谷氨酰胺类似物L-2-氨基-4-氧代-5-氯戊酸和6-重氮-5-氧代-L-正亮氨酸（DON）以及碘乙酰胺选择性地使依赖谷氨酰胺的活性失活。每亚基（分子量为57,000）掺入1当量的DON会导致依赖谷氨酰胺的活性完全失活，从而为每个单体有一个谷氨酰胺位点以及亚基的功能同一性提供了证据。用碘乙酰胺烷基化后，羧甲基半胱氨酸是从酸性水解物中分离出的唯一修饰氨基酸。依赖谷氨酰胺的活性对氧化敏感。通过与高浓度的二硫苏糖醇孵育可逆转暴露于空气中导致的失活。
