Altered cofactor binding affects stability and activity of human UDP-galactose 4'-epimerase: implications for type III galactosemia.

Abstract

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.

• Authors: Thomas J McCorvie, Ying Liu, Andrew Frazer, Tyler J Gleason, Judith L Fridovich-Keil, David J Timson
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1822 Issue 10 Pg. 1516-26 (Oct 2012) ISSN: 0006-3002 [Print] Netherlands
• PMID: 22613355 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• Coenzymes
• Fungal Proteins
• Recombinant Proteins
• NAD
• Uridine Diphosphate N-Acetylglucosamine
• Peptide Hydrolases
• UDPglucose 4-Epimerase
• Galactose

Topics

• Alleles
• Coenzymes (genetics, metabolism)
• Escherichia coli (genetics, metabolism)
• Fungal Proteins (metabolism)
• Galactose (genetics, metabolism)
• Galactosemias (enzymology, genetics)
• Humans
• Kinetics
• Models, Molecular
• NAD (genetics, metabolism)
• Peptide Hydrolases (genetics, metabolism)
• Protein Binding (genetics)
• Protein Denaturation
• Protein Multimerization
• Protein Structure, Secondary (genetics)
• Recombinant Proteins (genetics, metabolism)
• Structure-Activity Relationship
• UDPglucose 4-Epimerase (chemistry, genetics, metabolism)
• Uridine Diphosphate N-Acetylglucosamine (genetics, metabolism)
• Yeasts (genetics, metabolism)