A BIFUNCTIONAL ENZYME CATALYZES THE FIRST 2 STEPS IN N-ACETYLNEURAMINIC ACID BIOSYNTHESIS OF RAT-LIVER - PURIFICATION AND CHARACTERIZATION OF UDP-N-ACETYLGLUCOSAMINE 2-EPIMERASE N-ACETYLMANNOSAMINE KINASE/

Biosynthesis of N-acetylneuraminic acid (Neu5Ac), a prominent componen t of glycoconjugates, is initiated by the action of UDP-N-acetylglucos amine a-epimerase (UDP-GlcNAc a-epimerase, EC 5.1.3.14) and N-acetylma nnosamine kinase (ManNAc kinase, EC 2.7.1.60), We demonstrate for the first time that the two activities are parts of one bifunctional enzym e in rat liver, The enzyme was purified to homogeneity from rat liver cytosol using salmine sulfate precipitation and chromatography on phen yl-Sepharose, ATP-agarose, and Mono Q. The purification resulted in on e polypeptide with an apparent molecular mass of 75 kDa, Immunoprecipi tation with a polyclonal antibody against the polypeptide reduced both enzyme activities in equal amounts, Gel filtration analysis of purifi ed UDP-GlcNAc 2-epimerase/ManNAc kinase showed that the polypeptide se lf-associates as a dimer and as a hexamer with apparent molecular mass es of 150 and 450 kDa, respectively, The hexamer was fully active for both enzyme activities, whereas the dimer catalyzed only the phosphory lation of N-acetylmannosamine (ManNAc), Incubation of the dimer with U DP-N-acetylglucosamine led to reassembly of the fully active hexamer; maximal quantities of the hexamer were produced after incubation for 3 h. Kinetic analysis of purified hexameric and dimeric enzyme revealed significantly lower Michaelis constants (93 +/- 3 to 121 +/- 15 mu M for ManNAc and 1.18 +/- 0.13 to 1.67 +/- 0.20 mm for ATP) and higher c ooperativity (Hill coefficients of 1.42 +/- 0.16 to 1.17 +/- 0.06 for ManNAc and 1.30 +/- 0.09 to 1.05 +/- 0.14 for ATP) for the hexamer for both substrates of ManNAc kinase, The Michaelis constant of UDP-GlcNA c a-epimerase for its substrate was 11 +/- 2 mu M. The Hill coefficien t of 0.45 +/- 0,07 represents strongly negative cooperativity in subst rate binding, UDP-GlcNAc a-epimerase was feedback inhibited by CMP-Neu 5Ac. Complete inhibition was achieved with 60 mu M CMP-Neu5Ac, and hig hly positive cooperativity (Hill coefficient of 4.1) was found for inh ibitor binding.