1I0I

ANALYSIS OF AN INVARIANT ASPARTIC ACID IN HPRTS-GLUTAMINE MUTANT

1I0I の概要

• エントリーDOI: 10.2210/pdb1i0i/pdb
• 関連するPDBエントリー: 1I0L 1I13 1I14 1tc1 1tc2
• 分子名称: HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE, MAGNESIUM ION, 7-HYDROXY-PYRAZOLO[4,3-D]PYRIMIDINE, ... (5 entities in total)
• 機能のキーワード: phosphoribosyltransferase, nucleotide metabolism, purine salvage, ternary complex, catalytic base, transferase
• 由来する生物種: Trypanosoma cruzi
• 細胞内の位置: Cytoplasm : Q27796
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 52224.32

構造登録者

Canyuk, B.,Focia, P.J.,Eakin, A.E. (登録日: 2001-01-29, 公開日: 2002-05-29, 最終更新日: 2023-08-09)

主引用文献

Canyuk, B.,Focia, P.J.,Eakin, A.E.
The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.
Biochemistry, 40:2754-2765, 2001

PubMed Abstract: The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

PubMed: 11258886
DOI: 10.1021/bi001195q

実験手法

X-RAY DIFFRACTION (2.06 Å)