Metal ion activation of galactosyltransferase.
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+.
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
D002413:Cations, Divalent; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D007501:Iron; D005700:Galactosyltransferases; D015032:Zinc; D011221:Praseodymium