Substrate specificity of CTP: phosphoethanolamine cytidylyltransferase purified from rat liver.
CTP: phosphoethanolamine cytidylyltransferase was recently purified to homogeneity from rat liver (Vermeulen, P.S. Tijburg, L.B.M., Geelen, M.J.H. and van Golde, L.M.G. (1993) J. Biol. Chem. 268, 7458-7464). The present study focuses on the specificity of this enzyme for phosphorylated bases with a varying degree of N-methylation. The apparent Km for phosphoethanolamine was 0.072 mM. As the number of N-methylated substituents on phosphoethanolamine increased, the apparent Km increased: 0.11 mM for phosphomonomethylethanolamine and 6.8 mM for phosphodimethylethanolamine. Introduction of a third N-methyl group did not further increase the Km value. The effect of N-methyl groups on the reaction velocity was far more pronounced. A decreased Vmax for the reaction was found as the number of N-methyl substituents increased: 1.52 and 0.24 mumol/min per mg protein for phosphoethanolamine and phosphomonomethylethanolamine, and 44 and 0.69 nmol/min per mg protein for phosphodimethylethanolamine and phosphocholine, respectively. Phosphomonomethylethanolamine, phosphodimethylethanolamine and phosphocholine were weak competitive inhibitors of the cytidylyltransferase catalyzed reaction when phosphoethanolamine was used as a substrate, with Ki values of 7.0, 6.8 and 52.9 mM, respectively. The results show that this cytidylyltransferase is highly specific for phosphoethanolamine. Comparison of these data with previously reported information on the substrate specificity of CTP: phosphocholine cytidylyltransferase endorses the view that the two cytidylyltransferases are functionally different.
Vermeulen
P S
PS
Laboratory of Veterinary Biochemistry, Utrecht University, The Netherlands.
Geelen
M J
MJ
van Golde
L M
LM
D000818:Animals; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D012316:RNA Nucleotidyltransferases; D051381:Rats; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D009713:Nucleotidyltransferases; D012316:RNA Nucleotidyltransferases; C020126:Ethanolamine-phosphate cytidylyltransferase