Linked Life Data

9305887

Source:http://linkedlifedata.com/resource/pubmed/id/9305887

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
39
pubmed:dateCreated
1997-10-23
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
26
pubmed:volume
272
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
24313-8
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.
pubmed:affiliation
Institut für Molekularbiologie und Biochemie, Freie Universität Berlin, Arnimallee 22, D-14195 Berlin-Dahlem, Germany.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't