3VDA

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

Summary for 3VDA

• Entry DOI: 10.2210/pdb3vda/pdb
• Related: 1DP0 3VD3 3VD4 3VD5 3VD7 3VD9 3VDB 3VDC
• Descriptor: Beta-galactosidase, MAGNESIUM ION, SODIUM ION, ... (5 entities in total)
• Functional Keywords: transition state stabilization, substrate binding, 2-stage binding, tim barrel (alpha/beta barrel), jelly-roll barrel, immunoglobulin, beta supersandwich, glycosidase, hydrolase
• Biological source: Escherichia coli
• Total number of polymer chains: 4
• Total formula weight: 481847.63

Authors

Wheatley, R.W.,Kappelhoff, J.C.,Hahn, J.N.,Dugdale, M.L.,Dutkoski, M.J.,Tamman, S.D.,Fraser, M.E.,Huber, R.E. (deposition date: 2012-01-04, release date: 2012-04-11, Last modification date: 2023-09-13)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (2.5 Å)