Isolation and characterization of sialate lyase from pig kidney.
Sialate lyase (sialate aldolase; systematic name N-acetylneuraminate pyruvate-lyase, EC 4.1.3.3) was isolated as soluble enzyme from pig kidney and purified 630-fold using a heating step, gel filtration, and chromatography on immobilized neuraminic acid beta-methyl glycoside in 14% yield to apparent homogeneity as tested by SDS-gel electrophoresis. The molecular mass is 58 kDa and the pH-optimum is at pH 7.2. Kinetic parameters were determined with N-acetyl-neuraminic acid as substrate: Km 3.7 mM and Vmax 37.1 mU. The lyase cleaves only free sialic acids with relative rates of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for N-acetyl-9-O-acetylneuraminic acid, whereas N-acetyl-4-O-acetylneuraminic acid or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid are not substrates. Enzyme activity was inhibited with p-chloromercuribenzoate, o-phenanthroline, cyanide, 5-diazonium-1-H-tetrazole, 5,5'-dithiobis(2-nitrobenzoic acid), diethylpyro-carbonate, and Rose Bengal in the presence of light and O2. Reduction with sodium borohydride in the presence of N-acetylneuraminic acid or pyruvate resulted in irreversible inhibition of enzyme activity. The inhibition experiments suggest the involvement of histidine, lysine and SH-residues in enzyme catalysis. Thus, this mammalian lyase most probably belongs to the Class I aldolases, and has properties similar to the same enzyme from Clostridium perfringens and is active with the alpha-form of N-acetylneuraminic acid.
Schauer
R
R
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
Wember
M
M
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D009438:Neuraminic Acids / Q000737:chemistry / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D019163:Reducing Agents; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D000596:Amino Acids; D009438:Neuraminic Acids; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D006639:Histidine; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D008239:Lysine