Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activities of the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (E.C. 5.1.3 .14/2.7.1.60), the key enzyme of N-acetylneuraminic acid biosynthesis in ra t liver,,vas investigated. UDP-N-acetylglucosamine 2-epimerase was active i n imidazole/HCl buffer in the complete absence of any metal ion. 200 mM Na, K+, Rb+ and Cs+ activated enzyme activity up to five-fold, whereas lower concentrations of these monovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimerase activity. In sodium phosphate buffer t he enzyme activity was increased by 0.5 mM Mg2+, Sr2+, Ba2+ and Mn2+, while in the presence of 200 mM NaCl UDP-N-acetylglucosamine 2-epimerase activit y showed a stronger activation by these divalent metal ions. In imidazole/H Cl buffer, UDP-N-acetylglucosamine 2-epimerase activity was partially inhib ited by 0.5 mM Be2+, Mg2+, Ba2+, Mn2+, Sn2+ and Fe2+, and completely inhibi ted by 0.5 mM Zn2+ and Cd2+. Divalent metal ions were essential for N-acety lmannosamine kinase activity, the most effective being Mg2+, followed by Mn 2+ and Co2+. The optimal concentration of these metal ions was 3 mM. Less e ffective were Ni2+ and Cd2+, whereas Ca2+, Ba2+, Cu2+, Fe2+ and Zn2+ showed no effect on enzyme activity.