A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver

Stephan Hinderlich,Roger Stäsche,Reinhard Zeitler,Werner Reutter

doi:10.1074/jbc.272.39.24313

Stephan Hinderlich, Roger Stäsche + Show 2 more

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https://doi.org/10.1074/jbc.272.39.24313

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Abstract

Biosynthesis of N-acetylneuraminic acid (Neu5Ac), a prominent component of glycoconjugates, is initiated by the action of UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14) and N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60). We demonstrate for the first time that the two activities are parts of one bifunctional enzyme in rat liver. The enzyme was purified to homogeneity from rat liver cytosol using salmine sulfate precipitation and chromatography on phenyl-Sepharose, ATP-agarose, and Mono Q. The purification resulted in one polypeptide with an apparent molecular mass of 75 kDa. Immunoprecipitation with a polyclonal antibody against the polypeptide reduced both enzyme activities in equal amounts. Gel filtration analysis of purified UDP-GlcNAc 2-epimerase/ManNAc kinase showed that the polypeptide self-associates as a dimer and as a hexamer with apparent molecular masses of 150 and 450 kDa, respectively. The hexamer was fully active for both enzyme activities, whereas the dimer catalyzed only the phosphorylation of N-acetylmannosamine (ManNAc). Incubation of the dimer with UDP-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 microM for ManNAc and 1.18 +/- 0. 13 to 1.67 +/- 0.20 mM for ATP) and higher cooperativity (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-GlcNAc 2-epimerase for its substrate was 11 +/- 2 microM. The Hill coefficient of 0.45 +/- 0.07 represents strongly negative cooperativity in substrate binding. UDP-GlcNAc 2-epimerase was feedback inhibited by CMP-Neu5Ac. Complete inhibition was achieved with 60 microM CMP-Neu5Ac, and highly positive cooperativity (Hill coefficient of 4.1) was found for inhibitor binding.

Highlights

Biosynthesis of N-acetylneuraminic acid (Neu5Ac), a prominent component of glycoconjugates, is initiated by the action of UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1.3.14) and N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60)
UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1.3.14) catalyzes the formation of ManNAc from UDP-GlcNAc [2], and ManNAc is phosphorylated by N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) [3]
It has been suggested that UDP-GlcNAc 2-epimerase is the key enzyme of Neu5Ac biosynthesis because its activity is feedback inhibited by CMP-Neu5Ac [6, 8]

Summary

EXPERIMENTAL PROCEDURES

Salmine sulfate was from Roth (Karlsruhe, Germany). UDP-[U14C]GlcNAc and [1-14C]ManNAc were from ICN (Eschwege, Germany). All other chemicals were from Sigma (Deisenhofen, Germany) and Boehringer Mannheim (Mannheim, Germany). UDP-GlcNAc 2-epimerase and ManNAc kinase activities were assayed by a modified method of Zeitler et al [11]. The final volume of incubation mixtures was 225 ␮l, incubations were carried out at 37 °C for 30 min, and reactions were stopped by addition of 350 ␮l of ethanol. UDP-GlcNAc 2-epimerase assay: 45 mM Na2HPO4, pH 7.5, 10 mM MgCl2, 1 mM UDP-GlcNAc, 25 nCi of UDP-[14C]GlcNAc. ManNAc kinase assay: 60 mM Tris/HCl, pH 8.1, 20 mM MgCl2, 5 mM ManNAc, 50 nCi [14C]ManNAc, 20 mM ATP (disodium salt).

The abbreviations used are

RESULTS

DISCUSSION