Blakesley R W, Boezi J A
Biochim Biophys Acta. 1975 Dec 4;414(2):133-45. doi: 10.1016/0005-2787(75)90216-6.
A catalytic and structural study of ATP:RNA adenylyltransferase (EC 2.7.7.19) from the particulate fraction of Pseudomonas putida was made. During the large-scale purification of this enzyme, designated adenylyltransferase B, a previously undetected ATP-incorporating activity, designated adenylyltransferase A, was observed. Adenylyltransferases A and B were indistinguishable catalytically; however, they differed in their chromatographic and sedimentation properties. Adenylyltransferases A and B were resolved by phosphocellulose, by poly (U)-Sepharose and by Bio-Gel P-100 chromatographies. Adenylytransferase A was determined to have a sedimentation coefficient (S020,w) of 9.3 S and B of 4.3 S. The molecular weight of adenylyltransferase A was estimated to be 185000 and that of adenylyltransferase B to be 50000-60000. Apparently, adenylyltransferase A was generated from adenylyltransferase B during the purification. The AMP incorporation catalyzed by adenylyltransferases A and B was inhibited by two derivatives of the antibiotic rifamycin, AF/013 (50% at 5 mug/ml) and AF/DNFI (50% at 10 mug/ml). The 5'-triphosphate derivative (3'-dATP) of the drug cordycepin (3'-deoxyadenosine/ was a competitive inhibitor with ATP for both adenylyltransferases. The Ki for 3'-deoxyadenosine 5'-triphosphate was 6 - 10(-4)--10 - 10(-4) M, while the Km for ATP was 1 - 10(-4)--2 - 10(-4) M. Several other anaolgs of ATP, 2'-deoxyadenosine 5' triphosphate, 2'-O-methyl ATP, or the fluorescent 3-beta-D-ribofuranosylimidazo [2,1-i] purien 5'-triphosphate did not affect the activity of adenylyltransferase A or B. Poly(U) and poly(dT) were competitive inhibitors of the ribosomal RNA-primed polymerization reaction. The Ki for poly(U) or poly(dT), in terms of nucleotide phosphate, was 4 - 10-6)--10 - 10(-6) M for adenylyltransferases A and B, compared to 2 - 10(-4)--4 - 10(-4) M for the Km of ribosomal RNA. The inhibition was a result of the competition between the non-priming poly(U), or poly(dT), and ribosomal RNA for the primer binding site on the enzyme.
对恶臭假单胞菌颗粒部分的ATP：RNA腺苷酸转移酶（EC 2.7.7.19）进行了催化和结构研究。在大规模纯化这种被称为腺苷酸转移酶B的酶的过程中，观察到一种先前未检测到的掺入ATP的活性，称为腺苷酸转移酶A。腺苷酸转移酶A和B在催化方面无法区分；然而，它们在色谱和沉降特性上有所不同。腺苷酸转移酶A和B通过磷酸纤维素、聚（U）-琼脂糖和Bio-Gel P-100色谱法得以分离。测定腺苷酸转移酶A的沉降系数（S020,w）为9.3 S，B为4.3 S。腺苷酸转移酶A的分子量估计为185000，腺苷酸转移酶B的分子量为50000 - 60000。显然，腺苷酸转移酶A是在纯化过程中由腺苷酸转移酶B产生的。腺苷酸转移酶A和B催化的AMP掺入受到抗生素利福霉素的两种衍生物AF/013（5μg/ml时50%抑制）和AF/DNFI（10μg/ml时50%抑制）的抑制。药物虫草素（3'-脱氧腺苷）的5'-三磷酸衍生物（3'-dATP）是腺苷酸转移酶A和B与ATP竞争的竞争性抑制剂。3'-脱氧腺苷5'-三磷酸的Ki为6×10⁻⁴ - 1×10⁻⁴M，而ATP的Km为1×10⁻⁴ - 2×10⁻⁴M。其他几种ATP类似物，2'-脱氧腺苷5'三磷酸、2'-O-甲基ATP或荧光3-β-D-呋喃核糖基咪唑[2,1-i]嘌呤5'-三磷酸不影响腺苷酸转移酶A或B的活性。聚（U）和聚（dT）是核糖体RNA引发的聚合反应的竞争性抑制剂。腺苷酸转移酶A和B的聚（U）或聚（dT）的Ki（以核苷酸磷酸计）为4×10⁻⁶ - 1×10⁻⁶M，而核糖体RNA的Km为2×10⁻⁴ - 4×10⁻⁴M。这种抑制是由于非引发性聚（U）或聚（dT）与核糖体RNA竞争酶上的引物结合位点所致。
