1E1C

METHYLMALONYL-COA MUTASE H244A Mutant

1E1C の概要

• エントリーDOI: 10.2210/pdb1e1c/pdb
• 関連するPDBエントリー: 1REQ 2REQ 3REQ 4REQ 5REQ 6REQ 7REQ
• 分子名称: METHYLMALONYL-COA MUTASE ALPHA CHAIN, METHYLMALONYL-COA MUTASE BETA CHAIN, COBALAMIN, ... (5 entities in total)
• 機能のキーワード: isomerase, mutase, intramolecular transferase
• 由来する生物種: PROPIONIBACTERIUM FREUDENREICHII SUBSP. SHERMANII; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 303133.59

構造登録者

Evans, P.R.,Thoma, N.H. (登録日: 2000-04-30, 公開日: 2000-05-07, 最終更新日: 2023-12-06)

主引用文献

Thoma, N.H.,Evans, P.R.,Leadlay, P.F.
Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-Coa Mutase
Biochemistry, 39:9213-, 2000

PubMed Abstract: Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.

PubMed: 10924114
DOI: 10.1021/BI0004302

実験手法

X-RAY DIFFRACTION (2.62 Å)