Metal Activation of Galactosylhydroxylysyl Glucosyltransferase, an Intracellular Enzyme of Collagen Biosynthesis

Metal activation of galactosylhydroxylysyl glucosyltransferase was studied with EDTA-treated highly purified enzyme from chick embryos and EDTA-treated citrate-soluble collagen substrate. Mn2+ activated the transferase at much lower concentrations than thought previously, and high Mn2+ concentrations were more effective than could be expected from the Michaelis-Menten equation. The data suggest that the enzyme can bind at least two Mn2+ ions, the first having a Kd of 3–5 μM (site I) and the second 50–70 μM (site II). The binding of the second Mn2+ decreases the Km of UDP-glucose from 25 μM to 6–7 μM, whereas the V at a saturating concentration of UDP-glucose is unaffected. The EDTA-treated enzyme possessed some activity even without added Mn2+, suggesting that either the transferase can be activated in the absence of any metal or that it has one additional metal binding site with high affinity. The kinetic data would be consistent with a reaction scheme involving an ordered binding of Mn2+, UDP-glucose and the collagen substrate at low Mn2+ and an ordered binding of Mn2+, Mn2+, UDP-glucose and the collagen substrate at high Mn2+ concentrations. In both cases the binding of the Mn2+, which occurs before that of UDP-glucose, is at thermodynamic equilibrium. Zn2+, Cu2+, Cd2+, Ni2+ and Ca2+ are inhibitors of the Mn2+-activated enzyme. Fe2+ (Kd 5–7 μM) and Co2+ (Kd 30 μM) also activate the enzyme at low concentrations, whereas no further activation, but instead an inhibition, was found at high concentrations. Ca2+ and Mg2+, which have previously been reported to serve as metal co-factors, did not activate the enzyme alone and did not stimulate the glucosyltransferase activated at low Mn2+ concentrations. The Kd values for Mn2+ at site II and for Co2+ are probably much higher than the physiological concentrations, whereas the activation by Mn2+ at site I and by Fe2+ may be of physiological significance.