1px3
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E. COLI (LACZ) BETA-GALACTOSIDASE (G794A)
OverviewOverview
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.
About this StructureAbout this Structure
1PX3 is a Single protein structure of sequence from Escherichia coli with MG, NA and DMS as ligands. Active as Beta-galactosidase, with EC number 3.2.1.23 Full crystallographic information is available from OCA.
ReferenceReference
Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase., Juers DH, Hakda S, Matthews BW, Huber RE, Biochemistry. 2003 Nov 25;42(46):13505-11. PMID:14621996
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