Catalytic properties of the CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens: comparison with the mammalian enzyme.
The biosynthesis of N-glycolylneuraminic acid (Neu5Gc) was investigated in cell-free extracts of the starfish Asterias rubens, which is one of the evolutionarily least-advanced species known to possess Neu5Gc-containing glycoconjugates. As in higher animals, Neu5Gc is synthesised in Asterias rubens by the action of a CMP-Neu5Ac hydroxylase. Enzyme activity was detected in all starfish tissues tested, the turnover being the greatest in the gonads. The enzyme from this tissue has a temperature optimum between 25 and 33 degrees C and a pH optimum between pH 6.0 and 6.4. This hydroxylase exhibits many characteristics in common with the mammalian enzyme. For example, the enzyme is extracted in a predominantly soluble form. Oxygen and a reduced pyridine nucleotide are necessary for activity, with NADH being the most effective cofactor. Furthermore, the activation of the hydroxylase by exogenously added iron salts and the potent inhibitory effects of several iron ligands point to the involvement of a non-haem iron cofactor. The enzyme has a high affinity for the substrate CMP-Neu5Ac, the apparent Km being 18 microM. In contrast to the mammalian enzyme, the hydroxylase from Asterias rubens is not inhibited by increased ionic strength and cannot be activated by non-ionic detergents. Moreover, the CMP-Neu5Ac turnover increased linearily with increasing protein concentration. In accordance with other enzymes in starfish, seasonal changes in the CMP-Neu5Ac hydroxylase activity were also observed.
Schlenzka
W
W
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
Shaw
L
L
Schauer
R
R
D000818:Animals; D005075:Biological Evolution; D002248:Carbon Monoxide / Q000494:pharmacology; D002417:Cattle; D002855:Chromatography, Thin Layer; D003067:Coenzymes / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000378:metabolism*; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009994:Osmolar Concentration; D010100:Oxygen / Q000378:metabolism; D012621:Seasons; D013215:Starfish; D013696:Temperature
D003067:Coenzymes; D003902:Detergents; D008024:Ligands; D002248:Carbon Monoxide; D007501:Iron; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D003568:Cytidine Monophosphate; D010100:Oxygen