Characterization of acetyl-CoA synthetase kinetics and ATP-binding
| Autor: | Gema Lozano Terol, Manuel Cánovas Díaz, Teresa de Diego Puente, Barbara Zambelli, Julia Gallego-Jara, Ana Écija Conesa |
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| Přispěvatelé: | Department of Biochemistry and Molecular Biology and Immunology (B), Faculty of Chemistry, University of Murcia, Gallego-Jara J., Terol G.L., Ecija Conesa A., Zambelli B., Canovas Diaz M., de Diego Puente T. |
| Rok vydání: | 2019 |
| Předmět: |
Escherichia col
Lysine Biophysics Acetyl-CoA synthetase medicine.disease_cause Biochemistry 03 medical and health sciences Adenosine Triphosphate Acetyl Coenzyme A Escherichia coli medicine Enzyme kinetics lysine acetylation Molecular Biology Adenylylation 030304 developmental biology chemistry.chemical_classification 0303 health sciences 577 - Bioquímica. Biología molecular. Biofísica [- Biología] Chemistry Escherichia coli Proteins 030302 biochemistry & molecular biology Isothermal titration calorimetry Acetyl—CoA synthetase ITC ATP Kinetics Enzyme Acetylation Lysine acetylation Protein Binding |
| Zdroj: | DIGITUM. Depósito Digital Institucional de la Universidad de Murcia instname |
| ISSN: | 0304-4165 |
| DOI: | 10.1016/j.bbagen.2019.03.017 |
| Popis: | Background The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation. |
| Databáze: | OpenAIRE |
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