1S89

H98N Mutant of Methylglyoxal Synthase from E. coli complexed with Phosphoglycolic Acid

1S89 の概要

• エントリーDOI: 10.2210/pdb1s89/pdb
• 関連するPDBエントリー: 1S8A
• 分子名称: Methylglyoxal synthase, 2-PHOSPHOGLYCOLIC ACID (3 entities in total)
• 機能のキーワード: glycolytic bypass, methylglyoxal, lyase
• 由来する生物種: Escherichia coli
• 細胞内の位置: Cytoplasm: P0A731
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 102417.19

構造登録者

Marks, G.T.,Harrison, D.H.,Susler, M. (登録日: 2004-01-31, 公開日: 2004-03-30, 最終更新日: 2024-02-14)

主引用文献

Marks, G.T.,Susler, M.,Harrison, D.H.
Mutagenic studies on histidine 98 of methylglyoxal synthase: effects on mechanism and conformational change.
Biochemistry, 43:3802-3813, 2004

PubMed Abstract: Two detailed mechanisms [Marks et al. (2001) Biochemistry 40, 6805] have been proposed to explain the activity of methylglyoxal synthase (MGS), a homohexameric allosterically regulated enzyme that catalyzes the elimination of phosphate from DHAP to form enol pyruvaldehyde. This enol then tautomerizes to methylglyoxal in solution. In one of these mechanisms His 98 plays an obligate role in the transfer of a proton from the O(3) oxygen of DHAP to the O2 oxygen. To test this hypothesized mechanism, the variants H98N and H98Q were expressed and purified. Relative to the wild-type enzyme, the H98N variant shows a 50-fold decrease in k(cat) with all other kinetic parameters (i.e., K(m), K(PGA), etc.) being nearly the same. By contrast, the apparent catalytic rate for the H98Q variant is 250-fold lower than that of the wild-type enzyme. Inorganic phosphate acts as a competitive inhibitor (with a K(i) of 15 microM) rather than as an allosteric-type inhibitor as it does in the wild-type enzyme, and the competitive inhibitor phosphoglyolate (PGA) acts as an activator of this variant. These facts are explained by a shift in the conformational equilibrium toward an "inactive" state. When activation by PGA is accounted for, the catalytic rate for the "active" state of H98Q is estimated to be only 6-fold less than that of the wild-type enzyme, and thus His 98 is not essential for catalysis. Primary deuterium isotope effect data were measured for the wild-type enzyme and the two variants. At pH 7.0, the (D)V isotope effect (1.5) and the absence of a (D)(V/K) isotope effect for the wild-type enzyme suggest that the rate for the isotopically sensitive step is partially rate limiting but greater than the rate of substrate dissociation. Both the (D)V (2.0) and (D)(V/K) (3.4) isotope effects were more pronounced in the H98N variant, while the H98Q variant displayed an unusual inverse (D)V (0.8) isotope effect and a normal (D)(V/K) (1.5) isotope effect. The X-ray crystal structures of PGA bound to the H98Q and H98N variants were both determined to a resolution of 2.2 A. These mutations had little effect on the overall structure. However, the pattern of hydrogen bonding in the active site suggests an explanation as to how in the wild-type and H98N mutated enzymes the "inactive to active" equilibrium lies toward the active state, while with the H98Q mutated enzyme the equilibrium lies toward the inactive state. Thus, the role of His 98 appears to be more as a regulator of the enzyme's conformation rather than as a critical contributor to the catalytic mechanism.

PubMed: 15049687
DOI: 10.1021/bi035838o

実験手法

X-RAY DIFFRACTION (2.22 Å)