Some Kinetic Properties of Human-Milk Galactosyl Transferase

Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of ä-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of α-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order: Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 °C) the attachment of Mn2+ is not at thermo-dynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of α-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human α-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine α-lactalbumin with glucose, can be reasonably explained only by assuming that α-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that α-lactalbumin attaches to an enzyme Mn2+ UDP-galactose complex at thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of ä-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four α-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for α-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in α-lactalbumins.