2P6Z

Enzymatic and Structural Characterisation of Amphinase, a Novel Cytotoxic Ribonuclease from Rana pipiens Oocytes

2P6Z の概要

• エントリーDOI: 10.2210/pdb2p6z/pdb
• 関連するPDBエントリー: 2P7S
• 分子名称: Recombinant Amphinase-2, SODIUM ION, CITRIC ACID, ... (4 entities in total)
• 機能のキーワード: amphinase, cytotoxic rnase, enzyme efficiency, substrate specificity, hydrolase
• 由来する生物種: Rana pipiens (northern leopard frog)
• 細胞内の位置: Secreted : P85073
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 27121.57

構造登録者

Singh, U.P. (登録日: 2007-03-19, 公開日: 2007-05-29, 最終更新日: 2024-10-16)

主引用文献

Singh, U.P.,Ardelt, W.,Saxena, S.K.,Holloway, D.E.,Vidunas, E.,Lee, H.S.,Saxena, A.,Shogen, K.,Acharya, K.R.
Enzymatic and Structural Characterisation of Amphinase, a Novel Cytotoxic Ribonuclease from Rana pipiens Oocytes.
J.Mol.Biol., 371:93-111, 2007

PubMed Abstract: Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.

PubMed: 17560606
DOI: 10.1016/j.jmb.2007.04.071

実験手法

X-RAY DIFFRACTION (1.93 Å)