Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia

Area of Science:

• Biochemistry
• Enzymology
• Genetics

Background:

• A missense mutation (S135L) in human galactose 1-phosphate uridyltransferase (hGALT) is common in Black galactosemia patients.
• Homozygous S135L/S135L individuals exhibit no GALT activity in erythrocytes or lymphoblasts, yet show near-normal galactose oxidation.
• The biochemical basis for residual activity in leukocytes and overall metabolic compensation remains unclear.

Purpose of the Study:

• To elucidate the biochemical mechanisms underlying the S135L hGALT mutation's effects.
• To determine the catalytic role of serine at amino acid 135 and its contribution to protein stability.
• To differentiate between impaired catalysis and bio-instability caused by the leucine substitution.

Main Methods:

• Site-directed mutagenesis of hGALT at amino acid 135 was performed in a GALT-minus E. coli expression system.
• Enzyme activities of various hGALT mutants (S135, A135, C135, H135, L135, S132-H135, T135, Y135) were measured in bacterial lysates.
• Purified wild-type and L135-hGALT proteins were analyzed for kinetic parameters (Vmax, KM) and stability.

Main Results:

• Only the threonine substitution (S135T) retained significant enzyme activity in bacterial lysates.
• Mutant hGALT proteins showed decreased abundance in lysates due to proteolysis, indicating bio-instability.
• Purified L135-hGALT exhibited a significantly reduced Vmax (5.8 U/mg vs. 80 U/mg for wild-type) with no change in KM, suggesting impaired catalysis.

Conclusions:

• A hydroxyl group at amino acid 135 of hGALT is essential for efficient uridyl transfer catalysis.
• Amino acid 135 plays a critical role in the bio-stability of hGALT.
• The S135L mutation impairs both catalytic activity and protein stability, explaining observed galactosemia phenotypes.