PDBsum entry: 1px3

Hydrolase

PDB id: 1px3

Contents

Protein chains

1010 a.a. *

Ligands

DMS ×85

Metals

_MG ×12

_NA ×16

Waters ×3820

* Residue conservation analysis

PDB id:

1px3

Name:

Hydrolase

Title:

E. Coli (lacz) beta-galactosidase (g794a)

Structure:

Beta-galactosidase. Chain: a, b, c, d. Synonym: lactase. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: lacz. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PQS)

Resolution:

1.60Å R-factor: 0.192 R-free: 0.243

Authors:

D.H.Juers,S.Hakda,B.W.Matthews,R.E.Huber

Key ref:

D.H.Juers et al. (2003). Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase. Biochemistry, 42, 13505-13511. PubMed id: 14621996 DOI: 10.1021/bi035506j

Date:

02-Jul-03 Release date: 15-Jun-04

Protein chains

P00722  (BGAL_ECOLI) -  Beta-galactosidase

Seq: 1024 a.a.

Struc: 1010 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.2.1.23 - Beta-galactosidase.
• Reaction: Hydrolysis of terminal, non-reducing beta-D-galactose residues in beta-D-galactosides.

 Gene Ontology (GO) functional annotation 

• Cellular component: beta-galactosidase complex (1 term)
• Biological process: metabolic process (3 terms)
• Biochemical function: catalytic activity (9 terms)

DOI no: 10.1021/bi035506j

Biochemistry 42:13505-13511 (2003)

PubMed id: 14621996

Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase.

D.H.Juers, S.Hakda, B.W.Matthews, R.E.Huber.

ABSTRACT

The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor isopropyl thio-beta-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate constant for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.