Structural Basis for the Altered Activity of Gly794 Variants of Escherichia coli β-Galactosidase†
| Autor: | Reuben E. Huber, Douglas H. Juers, Shamina Hakda, Brian W. Matthews |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Protein Conformation Glycine Crystallography X-Ray medicine.disease_cause Binding Competitive Biochemistry Protein structure Hydrolase Escherichia coli medicine Enzyme kinetics Beta-galactosidase Enzyme Inhibitors Binding site Alanine Binding Sites biology Chemistry Methanol Active site beta-Galactosidase Molecular biology Recombinant Proteins Kinetics Glucose Amino Acid Substitution biology.protein Thermodynamics |
| Zdroj: | Biochemistry. 42:13505-13511 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi035506j |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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