A pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either alpha or beta-, but alpha- is somewhat preferred. In the absence of alpha-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of alpha-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (beta-configuration). However, upon alpha-lactalbumin binding the aglycone specificity allows for axial (alpha-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that alpha-lactalbumin binds to a hydrophobic site some distance from the C-2 group.

Glutathione (GSH) is an antioxidant essential for mammalian cell survival and is found in every cell of almost all organisms. GSH cannot be taken up by the cell, so understanding the intercellular synthesis of this important antioxidant is crucial. GSH is synthesized in a two‐step process. In the second step, human glutathione synthetase (hGS) binds γ‐glutamylcysteine and glycine and then uses the energy of ATP to make GSH. hGS is a homodimer that displays negative cooperativity, toward the γ‐glutamylcysteine substrate, a phenomenon which contributes to metabolic regulation but is not yet fully understood. The H‐loop (147‐TISASF‐152) of hGS is highly conserved and located near the binding site of γ‐glutamylcysteine. The role of H‐loop residues was examined using site‐directed mutagenesis of H‐loop residues, followed by purification and kinetic characterization of H‐loop mutant hGS enzymes. Our results show that the H‐loop mutant hGS enzymes have altered binding affinity (Km) for the negatively cooperative substrate, γ‐glutamylcysteine. Grant Funding Source: Supported by NIH R15GM086833 (MEA), REP Grant (TWU, MEA), Welch Chem Dept Grant (TWU)