SITE-DIRECTED MUTAGENESIS AND DELETION OF THE CARBOXYL TERMINUS OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE PROTEIN R2 - EFFECTS ON CATALYTIC ACTIVITY AND SUBUNIT INTERACTION
SITE-DIRECTED MUTAGENESIS AND DELETION OF THE CARBOXYL TERMINUS OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE PROTEIN R2 - EFFECTS ON CATALYTIC ACTIVITY AND SUBUNIT INTERACTION
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DOI：
10.1021/bi00135a009
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发表时间：
1992-05-26
期刊：
影响因子：
2.9
通讯作者：
HUANG, CY
中科院分区：
文献类型：
--
作者：
CLIMENT, I;SJOBERG, BM;HUANG, CY
大肠杆菌的核糖核苷酸还原酶由两个可分离的、不同的同源二聚体蛋白质组成，称为R1和R2。研究了R2的C-末端区域在形成R1 R2活性络合物中的作用。通过定点诱变设计具有全长多肽链和缺失最后30个羧基末端残基的截短的多肽链的异二聚体R2形式。与全长或截短的同源二聚体相比，这种蛋白质与R1的结合的动力学分析显示，R2的C-末端占其与R1的所有相互作用。异二聚体R2形式的固有解离常数，仅与R1（13 μ-M）接触一次，与先前获得的相同[Climent，I.，Sjoberg，B.- M.，和黄，C. Y.（1991）Biochemistry 30，5164-5171]，对于合成的C-末端肽，15-18-μ-M。我们还诱变的唯一两个不变的残基定位在C-末端区域的R2，谷氨酸-350和酪氨酸-356，丙氨酸。这些突变蛋白与R1的结合仍然紧密，但它们的催化活性受到严重影响。虽然E350 A蛋白表现出低活性（比野生型低240倍）但具有确定的活性，但Y356 A完全无活性。这些残基的催化作用，而不是结构的作用进行了讨论。
Ribonucleotide reductase from Escherichia coli consists of two dissociable, nonidentical homodimeric proteins called R1 and R2. The role of the C-terminal region of R2 in forming the R1R2 active complex has been studied. A heterodimeric R2 form with a full-length polypeptide chain and a truncated one missing the last 30 carboxyl-terminal residues was engineered by site-directed mutagenesis. Kinetic analysis of the binding of this protein to R1, compared with full-length or truncated homodimer, revealed that the C-terminal end of R2 accounts for all of its interactions with R1. The intrinsic dissociation constant of the heterodimeric R2 form, with only one contact to R1, 13-mu-M, is of the same magnitude as that obtained previously [Climent, I., Sjoberg, B.-M., & Huang, C. Y. (1991) Biochemistry 30, 5164-5171] for synthetic C-terminal peptides, 15-18-mu-M. We have also mutagenized the only two invariant residues localized at the C-terminal region of R2, glutamic acid-350 and tyrosine-356, to alanine. The binding of these mutant proteins to R1 remains tight, but their catalytic activity is severely affected. While E350A protein exhibits a low (240 times less active than the wild-type) but definitive activity, Y356A is completely inactive. A catalytic rather than structural role for these residues is discussed.