1FKX

MURINE ADENOSINE DEAMINASE (D296A)

1FKX の概要

• エントリーDOI: 10.2210/pdb1fkx/pdb
• 分子名称: ADENOSINE DEAMINASE, ZINC ION, 6-HYDROXY-1,6-DIHYDRO PURINE NUCLEOSIDE, ... (4 entities in total)
• 機能のキーワード: zinc cofactor, tim barrel, aminohydrolase
• 由来する生物種: Mus musculus (house mouse)
• 細胞内の位置: Cell membrane; Peripheral membrane protein; Extracellular side (By similarity): P03958
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 40007.84

構造登録者

Wilson, D.K.,Quiocho, F.A. (登録日: 1996-02-29, 公開日: 1996-08-01, 最終更新日: 2024-02-07)

主引用文献

Sideraki, V.,Mohamedali, K.A.,Wilson, D.K.,Chang, Z.,Kellems, R.E.,Quiocho, F.A.,Rudolph, F.B.
Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase.
Biochemistry, 35:7862-7872, 1996

PubMed Abstract: Two adjacent aspartates, Asp 295 and Asp 296, playing major roles in the reaction catalyzed by mouse adenosine deaminase (mADA) were altered using site-directed mutagenesis. These mutants were expressed and purified from an ADA-deficient bacterial strain and characterized. Circular dichroism spectroscopy shows the mutants to have unperturbed secondary structure. Their zinc content compares well to that of wild-type enzyme. Changing Asp 295 to a glutamate decreases the kcat but does not alter the Km for adenosine, confirming the importance of this residue in the catalytic process and its minimal role in substrate binding. The crystal structure of the D295E mutant reveals a displacement of the catalytic water from the active site due to the longer glutamate side chain, resulting in the mutant's inability to turn over the substrate. In contrast, Asp 296 mutants exhibit markedly increased Km values, establishing this residue's critical role in substrate binding. The Asp 296->Ala mutation causes a 70-fold increase in the Km for adenosine and retains 0.001% of the wild-type kcat/Km value, whereas the ASP 296->Asn mutant has a 10-fold higher Km and retains 1% of the wild-type kcat/Km value. The structure of the D296A mutant shows that the impaired binding of substrate is caused by the loss of a single hydrogen bond between a carboxylate oxygen and N7 of the purine ring. These results and others discussed below are in agreement with the postulated role of the adjacent aspartates in the catalytic mechanism for mADA.

PubMed: 8672487
DOI: 10.1021/bi952920d

実験手法

X-RAY DIFFRACTION (2.4 Å)