title: Metal ion activation of galactosyltransferase.
abstract: Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.
mesh_terms: D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002413:Cations, Divalent; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology; D003126:Colostrum / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008433:Mathematics; D008892:Milk / Q000201:enzymology; D011221:Praseodymium / Q000494:pharmacology; D011247:Pregnancy; D015032:Zinc / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D007501:Iron; D005700:Galactosyltransferases; D015032:Zinc; D011221:Praseodymium