3VDA

E. coli (lacZ) beta-galactosidase (N460T)

3VDA の概要

• エントリーDOI: 10.2210/pdb3vda/pdb
• 関連するPDBエントリー: 1DP0 3VD3 3VD4 3VD5 3VD7 3VD9 3VDB 3VDC
• 分子名称: Beta-galactosidase, MAGNESIUM ION, SODIUM ION, ... (5 entities in total)
• 機能のキーワード: transition state stabilization, substrate binding, 2-stage binding, tim barrel (alpha/beta barrel), jelly-roll barrel, immunoglobulin, beta supersandwich, glycosidase, hydrolase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 481847.63

構造登録者

Wheatley, R.W.,Kappelhoff, J.C.,Hahn, J.N.,Dugdale, M.L.,Dutkoski, M.J.,Tamman, S.D.,Fraser, M.E.,Huber, R.E. (登録日: 2012-01-04, 公開日: 2012-04-11, 最終更新日: 2023-09-13)

主引用文献

Wheatley, R.W.,Kappelhoff, J.C.,Hahn, J.N.,Dugdale, M.L.,Dutkoski, M.J.,Tamman, S.D.,Fraser, M.E.,Huber, R.E.
Substitution for Asn460 cripples {beta}-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.
Arch.Biochem.Biophys., 521:51-61, 2012

PubMed Abstract: Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site. Structural and kinetic studies were done with β-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower k(cat) values. Substrate affinity to N460D-β-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-β-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-β-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-β-galactosidase, the transition state is less stable due to an increased entropy penalty.

PubMed: 22446164
DOI: 10.1016/j.abb.2012.03.014

実験手法

X-RAY DIFFRACTION (2.5 Å)