Lactose synthase: effect of alpha-lactalbumin on substrate activity of N-acylglucosamines.
N-Acetyl-, N-propionyl-, N-butyryl- and N-valerylglucosamines were synthesized as topographical probes to localize further the interaction site of alpha-lactalbumin on galactosyltransferase. All these compounds were found to be substrates for galactosyltransferase with Km values in the millimolar range. In the presence of alpha-lactalbumin, the Michaelis-Menten constants were diminished. However, the effect on the initial rates of these reactions varied. Thus, at low N-acylglucosamine concentrations, alpha-lactalbumin activated the enzyme activity, but at high concentrations, alpha-lactalbumin became inhibitory. This mixed-type inhibition kinetics indicated that a quaternary complex between galactosyltransferase, alpha-lactalbumin, Mn2+-UDPgalactose and N-acylglucosamine existed during the catalytic process. The ability of these N-acylglucosamine substrates to bind to lactose synthase complex was further substantiated by the physical association of galactosyltransferase onto the solid-bound alpha-lactalbumin in the presence of any one of these compounds. The data revealed that the presence of the N-acyl group up to five carbons in length did not interfere with the interaction between alpha-lactalbumin and galactosyltransferase, suggesting that alpha-lactalbumin was not bound in the vicinity of the C-2 region of the monosaccharide site. The inhibitory effect of alpha-lactalbumin on N-acyllactosamine formation is probably a consequence of conformational changes of galactosyltransferase.
D000818:Animals; D002417:Cattle; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives*; D007700:Kinetics; D007768:Lactalbumin / Q000494:pharmacology*; D007788:Lactose Synthase / Q000378:metabolism*; D008892:Milk / Q000201:enzymology; D011485:Protein Binding; D013379:Substrate Specificity
D007768:Lactalbumin; D005700:Galactosyltransferases; D007788:Lactose Synthase; D005944:Glucosamine