Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia
| Autor: | Judith L. Fridovich-Keil, Tyler J. Gleason, David J. Timson, Ying Liu, Thomas J. McCorvie, Andrew Frazer |
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| Jazyk: | angličtina |
| Předmět: |
Galactosemias
Models Molecular Protein Denaturation UDP-galactose 4′-epimerase Coenzymes Type III galactosemia Biology Cofactor Article Protein Structure Secondary Fungal Proteins 03 medical and health sciences Structure-Activity Relationship UDPglucose 4-Epimerase Protein structure Yeasts medicine Escherichia coli Humans Disease-associated mutation Molecular Biology Alleles 030304 developmental biology Yeast model chemistry.chemical_classification 0303 health sciences Cofactor binding Uridine Diphosphate N-Acetylglucosamine 030302 biochemistry & molecular biology Galactosemia Galactose medicine.disease NAD Molecular biology Yeast Recombinant Proteins GALE Kinetics Enzyme chemistry Biochemistry Differential scanning fluorimetry biology.protein Molecular Medicine Salt bridge NAD+ kinase Protein Multimerization Peptide Hydrolases Protein Binding |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. (10):1516-1526 |
| ISSN: | 0925-4439 |
| DOI: | 10.1016/j.bbadis.2012.05.007 |
| Popis: | Deficiency of UDP-galactose 4′-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD+. p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD+. These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation. |
| Databáze: | OpenAIRE |
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