The role of alpha-lactalbumin and the A protein in lactose synthetase: a unique mechanism for the control of a biological reaction.

Abstract

Lactose synthetase catalyzes the final step in the biosynthesis of lactose in the mammary gland by the reaction UDP-D-galactose + D-glucose --lactose + UDP.(1) A soluble, partially purified form of the enzyme, found in bovine milk, can be separated by gel filtration into two protein components, designated the A and B proteins. Neither component will catalyze reaction (1) separately, but when they are combined, lactose synthesis is obtained. More recently, the B protein has been found to be identical with the familiar milk protein, a-lactalbumin.2 3 Work in our laboratory has shown that the amino acid sequence of bovine a-LA is very similar to that of hen egg-white lysozyme.4 This homology in primary structure suggests that the structural genes for lysozyme and a-LA have evolved from a relatively recent, common ancestor. The structural similarities also suggest that a-LA may have a conformation quite similar to that established for lysozyme.A It is possible to fit the side chains of bovine a-LA to the lysozyme polypeptide backbone, and thereby generate a structure which retains the major structural features of the lysozyme molecule.6The studies reported here have been designed to define the roles of the A protein and a-LA in lactose synthetase. The separated A protein has been found to be a UDP-galactose: N-acetylglucosamine galactosyltransferase which catalyzes the following reaction: UDP-galactose + N-acetyl-D-glucosamine-N-acetyllactosamine + UDP. (2) Under normal assay conditions, a-LA inhibits this reaction and allows synthesis of lactose in the presence of glucose by reaction (1). Thus, the a-LA modifies the substrate (acceptor) specificity of a galactosyltransferase from NAG to glucose. This appears to be a role hitherto not ascribed to a protein and a-LA may be termed a "specifier" protein. The mechanism by which it effects a change in substrate specificity appears to be complex, but it is possible that it represents a new type of molecular control of a biological reaction. Experimental Methods.-Assay of UDP-galactose: glucose (31--*4 galactosyltransferase (lactose synthetase) and UDP-galactose: N-acetylglucosamine (1--*4 galactosyltransferase (N-acetyllactosamine [NAL]synthetase): Assays were performed by a modification of the method of Babad and Hassid.' NAL synthetase was determined by incubating 100-jiliter mixtures containing 5 Mmoles Tris-HCl, pH 7.4, 4 Mmoles MnC12, 2 /Amoles NAG, 63 m/Amoles [C14] UDP-galactose (10,000 cpm) (Calbiochem), and enzyme for 5-20 min at 37CC. Appropriate controls were included to correct for the small amount of nonspecific hydrolysis of UDP-galactose.

Amino acid sequence and crystal structure of buffalo α‐lactalbumin

“…In the presence of c~-LA, the Km of GT for glucose is reduced by up to 3 orders of magnitude so that lactose synthesis proceeds effectively at physiological levels of glucose, cx-LA, a major protein of the whey fraction of milk, possesses a number of biological features in addition to its role as a 'specifier' protein in the LS complex. Even though functionally divergent, it is homologous to C-type lysozymes [2]. It is a calcium binding protein [3] and binds different metal ions [4,5].…”

Characterization of Rhodosaminyl Transfer by the AknS/AknT Glycosylation Complex and Its Use in Reconstituting the Biosynthetic Pathway of Aclacinomycin A

“…This Gtf, β1,4-Galactosytransferase-1 (β4-Gal-T1), interacts with α-lactalbumin(LA). 15 The binding interaction stabilizes a particular conformation of Gal-T1 such that it accelerates the rate of transfer of sugars that are otherwise poor substrates. 16 Detailed kinetic analysis of this system have revealed substantial changes (1000 fold) in the K m of Gal-T1 for glucose in the presence or absence of LA.…”

The effect of temperature on a reaction catalysed by lactose synthetaseA protein

“…2a). On the other hand, at higher temperatures and low NAG concentrations the activity of the human milk enzyme, like that of the cow's milk enzyme at 30 ° [3] and 37 ° [1,2], is greater in the presence than in the absence of a.-lactalbumin, with the difference being more pro, nounced at 37 ° than at 30 ° (figs. 2b, 2c).…”

“…Addition ofct-lactalbumin lowers the K m for both glucose and NAG and greatly enhances the first reaction, but it may either enhance or inhibit the second reaction. It activates the enzyme from cow's milk at low NAG concentrations and inhibits it at high concentrations [1][2][3], whereas it inhibits the enzyme from human milk at all NAG concentrations [4]. These differences have now been reconciled and explained by taking into account the effect of temperature on the reaction.…”