Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver.
Sialic acids are a group of carboxylated amino sugars important for a variety of cellular functions. N-Acetylneuraminic acid (Neu5Ac) is the predominant sialic acid in nature. Neu5Ac-9-phosphate synthase catalyzes the formation of Neu5Ac-9-phosphate from N-acetylmannosamine-6-phosphate and phosphoenolpyruvate. Neu5Ac-9-phosphate synthase was purified 11,700-fold from rat liver cytosol to apparent homogeneity by ammonium sulfate precipitation, chromatography on hydroxylapatite, phenyl-Sepharose, MonoQ, and finally gel filtration. SDS-PAGE and gel filtration chromatography indicated that the enzyme is a dimer composed of 37-kDa subunits. Analysis of trypic peptides by MALDI-TOF MS verified a high sequence similarity to the corresponding murine enzyme. The K(m) values of Neu5Ac-9-phosphate synthase were 35 microM for N-acetylmannosamine-6-phosphate and 100 microM for phosphoenolpyruvate. The enzyme displayed an absolute requirement for divalent cations, Mn(2+), Fe(2+), and Mg(2+) being the most effective. In contrast to human Neu5Ac-9-phosphate synthase, the rat enzyme did not utilize mannose-6-phosphate in the synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid 9-phosphate. Neu5Ac-9-phosphate synthase was inactivated by the sulfhydryl modifying reagents, 5,5'-dithio-bis (2-nitrobenzoic acid) and N-ethylmaleimide, and protected from inactivation by the presence of the substrate phosphoenolpyruvate, but not by the presence of N-acetylmannosamine-6-phosphate, showing that at least one cysteine residue is located in the active site of the enzyme.
Chen
Hao
H
Institute of Molecular Biology and Biochemistry, Free University of Berlin, Arnimallee 22, 14195 Berlin-Dahlem, Germany.
Blume
Astrid
A
Zimmermann-Kordmann
Martin
M
Reutter
Werner
W
Hinderlich
Stephan
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; C018767:N-acylneuraminate-9-phosphate synthase; D014357:Trypsin; D007652:Oxo-Acid-Lyases; D003545:Cysteine