Identification of lysine 122 and arginine 196 as important functional residues of rat CTP:Phosphocholine cytidylyltransferase alpha

CTP:phosphocholine cytidylyltransferase (alpha (CCTalpha) contains a central region that functions as a catalytic domain, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the subsequent synthesis of phosphatidylcholine. We have investigated the catalytic role of lysine 122 and arginine 196 of rat CCTalpha using site-directed mutagenesis and a baculovirus expression system. Arginine 196 is part of the highly conserved RTEGIST motif, while lysine 122 has not previously been identified by protein sequence alignment as a candidate catalytic amino acid. Removing the side chain of lysine 122 compromises the catalytic ability of CCTalpha, decreasing the apparent V-max value in mutant enzymes Lys122Ala and Lys122Arg to 0.30 and 0.09% of the wild-type value, respectively. The decrease in V-max, is accompanied by dramatic 471 - and 80-fold increases in the apparent K-m value for phosphocholine but no greater than 3-fold increases in the apparent Hill constant (K*) value for CTP. Mutation of arginine 196 to lysine results in an enzyme that retains 24% of the wild-type V-max value with a modest 5-fold increase in the Km value for phosphocholine. However, the Arg196Lys mutant enzyme exhibits a 23-fold increase in the K* value for CTP. These data suggest lysine 122 and arginine 196 of rat CTP:phosphocholine cytidylyltransferase are functionally important amino acids, perhaps at or near the active site involved in forming contacts with the substrates phosphocholine and CTP, respectively.