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). T he present study focuses on the specificity of this enzyme for phospho rylated bases with a varying degree of N-methylation. The apparent K(m ) for phosphoethanolamine was 0.072 mM. As the number of N-methylated substituents on phosphoethanolamine increased, the apparent K(m) incre ased: 0.11 mM for phosphomonomethylethanolamine and 6.8 mM for phospho dimethylethanolamine. Introduction of a third N-methyl group did not f urther increase the K(m) value. The effect of N-methyl groups on the r eaction velocity was far more pronounced. A decreased V(max) for the r eaction was found as the number of N-methyl substituents increased: 1. 52 and 0.24 mumol/min per mg protein for phosphoethanolamine and phosp homonomethylethanolamine, and 44 and 0.69 nmol/min per mg protein for phosphodimethylethanolamine and phosphocholine, respectively. Phosphom onomethylethanolamine, phosphodimethylethanolamine and phosphocholine were weak competitive inhibitors of the cytidylyltransferase catalyzed reaction when phosphoethanolamine was used as a substrate, with K(i) values of 7.0, 6.8 and 52.9 mM, respectively. The results show that th is cytidylyltransferase is highly specific for phosphoethanolamine. Co mparison of these data with previously reported information on the sub strate specificity of CTP: phosphocholine cytidylyltransferase endorse s the view that the two cytidylyltransferases are functionally differe nt.