Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• UDP-galactose 4 -epimerase (GALE) is crucial for the Leloir pathway, interconverting UDP-galactose and UDP-glucose.
• Human GALE (hGALE) exhibits broader substrate specificity than bacterial GALE, also processing UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine.
• Previous X-ray crystallography data suggested active site volume influences hGALE substrate selectivity.

Purpose of the Study:

• To directly test the hypothesis that active site cleft volume dictates hGALE substrate specificity.
• To investigate the role of specific amino acid residues in hGALE's differential substrate recognition.

Main Methods:

• Generated four mutant hGALE alleles (S132A, Y157F, S132A/Y157F, C307Y) to probe active site function.
• Expressed and characterized mutant hGALE activity with UDP-galactose and UDP-N-acetylgalactosamine in Saccharomyces cerevisiae.
• Purified C307Y and S132A/Y157F-hGALE proteins from Pichia pastoris for detailed kinetic analysis.

Main Results:

• Mutations S132A and Y157F, individually or combined, abolished hGALE activity for both substrates.
• The C307Y mutation resulted in normal UDP-galactose epimerase activity but complete loss of UDP-N-acetylgalactosamine activity.
• These findings indicate that residue 307 is critical for accommodating the larger substrate.

Conclusions:

• The results validate the crystal structure of wild-type hGALE.
• Residue 307 functions as a gatekeeper, controlling access of larger substrates to the hGALE active site.
• Differential active site volume is a key determinant of hGALE's substrate selectivity.