3MUY

E. coli (lacZ) beta-galactosidase (R599A)

Summary for 3MUY

• Entry DOI: 10.2210/pdb3muy/pdb
• Related: 1DP0 1JYX 1JZ5 1PX3 1PX4 3MUZ 3MV0 3MV1
• Descriptor: Beta-D-galactosidase, DIMETHYL SULFOXIDE, MAGNESIUM ION, ... (5 entities in total)
• Functional Keywords: arg-599-ala, beta-galactosidase, hydrolase, tim barrel(alpha/beta barrel), jelly-roll barrel, immunoglobulin beta supersandwhich, glycosidase
• Biological source: Escherichia coli
• Total number of polymer chains: 4
• Total formula weight: 476448.80

Authors

Dugdale, M.L.,Vance, M.,Driedger, M.L.,Nibber, A.,Tran, A.,Huber, R.E. (deposition date: 2010-05-03, release date: 2011-03-16, Last modification date: 2023-09-06)

Primary citation

Dugdale, M.L.,Vance, M.L.,Wheatley, R.W.,Driedger, M.R.,Nibber, A.,Tran, A.,Huber, R.E.
Importance of Arg-599 of beta-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop
Biochem.Cell Biol., 88:969-979, 2010

PubMed Abstract: Structural and kinetic data show that Arg-599 of β-galactosidase plays an important role in anchoring the "open" conformations of both Phe-601 and an active-site loop (residues 794-803). When alanine was substituted for Arg-599, the conformations of Phe-601 and the loop shifted towards the "closed" positions because interactions with the guanidinium side chain were lost. Also, Phe-601, the loop, and Na+, which is ligated by the backbone carbonyl of Phe-601, lost structural order, as indicated by large B-factors. IPTG, a substrate analog, restored the conformations of Phe-601 and the loop of R599A-β-galactosidase to the open state found with IPTG-complexed native enzyme and partially reinstated order. ᴅ-Galactonolactone, a transition state analog, restored the closed conformations of R599A-β-galactosidase to those found with ᴅ-galactonolactone-complexed native enzyme and completely re-established the order. Substrates and substrate analogs bound R599A-β-galactosidase with less affinity because the closed conformation does not allow substrate binding and extra energy is required for Phe-601 and the loop to open. In contrast, transition state analog binding, which occurs best when the loop is closed, was several-fold better. The higher energy level of the enzyme•substrate complex and the lower energy level of the first transition state means that less activation energy is needed to form the first transition state and thus the rate of the first catalytic step (k2) increased substantially. The rate of the second catalytic step (k3) decreased, likely because the covalent form is more stabilized than the second transition state when Phe-601 and the loop are closed. The importance of the guanidinium group of Arg-599 was confirmed by restoration of conformation, order, and activity by guanidinium ions.

PubMed: 21102659
DOI: 10.1139/O10-144

Experimental method

X-RAY DIFFRACTION (2.5 Å)