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
4TTG 2014-06-20T00:00:00+0000 1.6 Beta-galactosidase (E. coli) in the presence of potassium chloride.
1DP0 1999-12-22T00:00:00+0000 1.7 E. COLI BETA-GALACTOSIDASE AT 1.7 ANGSTROM
1F4A 2000-06-07T00:00:00+0000 2.8 E. COLI (LACZ) BETA-GALACTOSIDASE (NCS CONSTRAINED MONOMER-ORTHORHOMBIC)
1F4H 2000-06-07T00:00:00+0000 2.8 E. COLI (LACZ) BETA-GALACTOSIDASE (ORTHORHOMBIC)
1HN1 2000-12-05T00:00:00+0000 3.0 E. COLI (LAC Z) BETA-GALACTOSIDASE (ORTHORHOMBIC)
1JYN 2001-09-13T00:00:00+0000 1.8 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH LACTOSE
1JYV 2001-09-13T00:00:00+0000 1.75 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH ONPG
1JYW 2001-09-13T00:00:00+0000 1.55 E. COLI (lacZ) BETA-GALACTOSIDASE (E537Q) IN COMPLEX WITH PNPG
1JYX 2001-09-13T00:00:00+0000 1.75 E. COLI (lacZ) BETA-GALACTOSIDASE IN COMPLEX WITH IPTG
1JZ2 2001-09-13T00:00:00+0000 2.1 E. COLI (lacZ) BETA-GALACTOSIDASE-TRAPPED 2-F-GALACTOSYL-ENZYME INTERMEDIATE (ORTHORHOMBIC)

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