Thermal denaturation of beta-galactosidase and of two site-specific mutants.


Abstract

The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion. Study holds ProTherm entries: 4351, 4352, 4353 Extra Details: additive : EDTA(1 mM), active site; beta-sheet; secondary structure; ligand binding;,ligand-free enzyme

Submission Details

ID: ehgJF8k83

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Edwards RA;Jacobson AL;Huber RE,Biochemistry (1990) Thermal denaturation of beta-galactosidase and of two site-specific mutants. PMID:2125499
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1JZ8 2001-12-07 1.5 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH ALLOLACTOSE
1JZ7 2001-12-07 1.5 E. COLI (lacZ) BETA-GALACTOSIDASE IN COMPLEX WITH GALACTOSE
1JYW 2001-12-07 1.55 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH PNPG
4TTG 2015-03-25 1.6 Beta-galactosidase (E. coli) in the presence of potassium chloride.
1PX4 2004-06-15 1.6 E. COLI (LACZ) BETA-GALACTOSIDASE (G794A) WITH IPTG BOUND
1PX3 2004-06-15 1.6 E. COLI (LACZ) BETA-GALACTOSIDASE (G794A)
1DP0 2001-02-21 1.7 E. COLI BETA-GALACTOSIDASE AT 1.7 ANGSTROM
1JYX 2001-12-07 1.75 E. COLI (lacZ) BETA-GALACTOSIDASE IN COMPLEX WITH IPTG
3T09 2012-01-18 1.75 E. coli (LacZ) beta-galactosidase (S796A) galactonolactone complex
3DYP 2008-10-28 1.75 E. coli (lacZ) beta-galactosidase (H418N)
1JYV 2001-12-07 1.75 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH ONPG
1JZ3 2001-12-07 1.75 E. COLI (lacZ) BETA-GALACTOSIDASE-TRAPPED 2-DEOXY-GALACTOSYL ENZYME INTERMEDIATE
1JYN 2001-12-07 1.8 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH LACTOSE
1JZ5 2001-12-07 1.8 E. COLI (lacZ) BETA-GALACTOSIDASE IN COMPLEX WITH D-GALCTOPYRANOSYL-1-ON
3DYO 2008-10-28 1.8 E. coli (lacZ) beta-galactosidase (H418N) in complex with IPTG
3T2O 2012-01-18 1.85 E. coli (lacZ) beta-galactosidase (S796D)
3T0A 2012-01-18 1.9 E. coli (LacZ) beta-galactosidase (S796T)
3MUZ 2011-03-16 1.9 E.Coli (lacZ) beta-galactosidase (R599A) in complex with IPTG
6CVM 2018-05-30 1.9 Atomic resolution cryo-EM structure of beta-galactosidase
3T0D 2012-01-18 1.93 E.coli (lacZ) beta-galactosidase (S796T) in complex with galactonolactone
4CKD 2014-01-15 13.0 Model of complex between the E.coli enzyme beta-galactosidase and four single chain Fv antibody domains scFv13R4.
3VD4 2012-04-11 2.0 E. coli (lacZ) beta-galactosidase (N460D) in complex with IPTG
3IAP 2009-12-29 2.0 E. coli (lacZ) beta-galactosidase (E416Q)
3T08 2012-01-18 2.0 E. coli (LacZ) beta-galactosidase (S796A) IPTG complex
3SEP 2012-01-18 2.05 E. coli (lacZ) beta-galactosidase (S796A)
3DYM 2008-10-28 2.05 E. coli (lacZ) beta-galactosidase (H418E)
3CZJ 2009-04-07 2.05 E. COLI (lacZ) BETA-GALACTOSIDASE (N460T) IN COMPLEX WITH D-GALCTOPYRANOSYL-1-ONE
3VDB 2012-04-11 2.05 E. coli (lacZ) beta-galactosidase (N460T) in complex with galactonolactone
3VD9 2012-04-11 2.05 E. coli (lacZ) beta-galactosidase (N460S) in complex with IPTG
3I3E 2010-05-12 2.1 E. COLI (lacZ) BETA-GALACTOSIDASE (M542A)
1JZ2 2001-12-07 2.1 E. COLI (lacZ) BETA-GALACTOSIDASE-TRAPPED 2-F-GALACTOSYL-ENZYME INTERMEDIATE (ORTHORHOMBIC)
1JZ6 2001-12-07 2.1 E. COLI (lacZ) BETA-GALACTOSIDASE IN COMPLEX WITH GALACTO-TETRAZOLE
1JZ4 2001-12-07 2.1 E. COLI (lacZ) BETA-GALACTOSIDASE-TRAPPED 2-DEOXY-GALACTOSYL-ENZYME INTERMEDIATE (Low Bis-Tris)
4DUV 2013-04-03 2.1 E. coli (lacZ) beta-galactosidase (G974A) 2-deoxy-galactosyl-enzyme and bis-Tris complex
3MV1 2011-03-16 2.2 E.Coli (lacZ) beta-galactosidase (R599A) in complex with Guanidinium
5A1A 2015-05-06 2.2 2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor
3MV0 2011-03-16 2.2 E. COLI (lacZ) BETA-GALACTOSIDASE (R599A) IN COMPLEX WITH D-GALCTOPYRANOSYL-1-ONE
6DRV 2018-07-11 2.2 Beta-galactosidase
3I3B 2010-05-12 2.2 E.coli (lacz) Beta-Galactosidase (M542A) in Complex with D-Galactopyranosyl-1-on
4DUW 2013-03-20 2.2 E. coli (lacZ) beta-galactosidase (G974A) in complex with allolactose
3I3D 2010-05-12 2.2 E. COLI (lacZ) BETA-GALACTOSIDASE (M542A) IN COMPLEX WITH IPTG
4DUX 2013-03-20 2.3 E. coli (lacZ) beta-galactosidase (N460S) in complex with L-ribose
3T2Q 2012-01-18 2.4 E. coli (lacZ) beta-galactosidase (S796D) in complex with galactonolactone
3T0B 2012-01-18 2.4 E. coli (LacZ) beta-galactosidase (S796T) IPTG complex
4V40 2014-07-09 2.5 BETA-GALACTOSIDASE
3VDA 2012-04-11 2.5 E. coli (lacZ) beta-galactosidase (N460T)
3MUY 2011-03-16 2.5 E. coli (lacZ) beta-galactosidase (R599A)
4V41 2014-07-09 2.5 E. COLI (LAC Z) BETA-GALACTOSIDASE (NCS CONSTRAINED MONOMER-MONOCLINIC)
3VDC 2012-04-11 2.55 E. coli (lacZ) beta-galactosidase (N460T) in complex with IPTG
3T2P 2012-01-18 2.6 E. coli (lacZ) beta-galactosidase (S796D) in complex with IPTG
4V45 2014-07-09 2.6 E. COLI (lacZ) BETA-GALACTOSIDASE-TRAPPED 2-F-GALACTOSYL-ENZYME INTERMEDIATE
3VD5 2012-04-11 2.7 E. coli (lacZ) beta-galactosidase (N460S)
3IAQ 2009-12-29 2.7 E. coli (lacz) beta-galactosidase (E416V)
4V44 2014-07-09 2.7 E. COLI (lacZ) BETA-GALACTOSIDASE IN COMPLEX WITH 2-F-LACTOSE
1F4A 2001-02-21 2.8 E. COLI (LACZ) BETA-GALACTOSIDASE (NCS CONSTRAINED MONOMER-ORTHORHOMBIC)
1F4H 2001-02-21 2.8 E. COLI (LACZ) BETA-GALACTOSIDASE (ORTHORHOMBIC)
3VD3 2012-04-11 2.8 E. coli (lacZ) beta-galactosidase (N460D)
3VD7 2012-04-11 2.87 E. coli (lacZ) beta-galactosidase (N460S) in complex with galactotetrazole
1HN1 2001-12-12 3.0 E. COLI (LAC Z) BETA-GALACTOSIDASE (ORTHORHOMBIC)
3E1F 2009-06-09 3.0 E.Coli (lacZ) beta-galactosidase (H418E) in complex with galactose
3J7H 2014-07-30 3.2 Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.8 Beta-galactosidase Q32JB6 BGAL_SHIDS
96.8 Beta-galactosidase A7KGA5 BGAL2_KLEPN
96.7 Beta-galactosidase A6TI29 BGAL2_KLEP7
97.3 Beta-galactosidase B5Z2P7 BGAL_ECO5E
97.4 Beta-galactosidase Q8X685 BGAL_ECO57
97.3 Beta-galactosidase B1LIM9 BGAL_ECOSM
97.5 Beta-galactosidase A1A831 BGAL_ECOK1
97.5 Beta-galactosidase Q8FKG6 BGAL_ECOL6
97.6 Beta-galactosidase Q1RFJ2 BGAL_ECOUT
97.6 Beta-galactosidase Q0TKT1 BGAL_ECOL5
97.9 Beta-galactosidase Q3Z583 BGAL_SHISS
98.0 Beta-galactosidase B7UJI9 BGAL_ECO27
98.0 Beta-galactosidase B7N8Q1 BGAL_ECOLU
99.0 Beta-galactosidase A7ZI91 BGAL_ECO24
99.0 Beta-galactosidase A7ZWZ1 BGAL_ECOHS
99.4 Beta-galactosidase Q8VNN2 BGAL_ECOLX
99.8 Beta-galactosidase B1J0T5 BGAL_ECOLC
100.0 Beta-galactosidase P00722 BGAL_ECOLI