Catalytic Roles of Conserved Aspartates of CTP:Phosphocholine Cytidylyltransferase

Abstract

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is a member of the CDP-choline pathway for the biosynthesis of phosphatidylcholine. Rat CCT alpha contains an N-terminal nuclear localization signal, a central catalytic portion, and C-terminal lipid binding and phosphorylation regions. We have investigated the catalytic role of conserved residues aspartate 86, aspartate 115, aspartate 143, and aspartate 164 of the catalytic domain of rat CCT using site-directed mutagenesis. Each aspartate was substituted by alanine and the enzymes were expressed in and purified from E.coli. Upon expression in bacterial cells at 37 degrees mutant enzymes D86A, D143A, and D164A were insoluble and present in inclusion bodies. Solubilization and purification of these mutant enzymes was accomplished by either expression at 10 degrees or denaturation / renaturation of the insoluble form. Upon mutation of aspartate 86 to alanine the ability of the mutant enzyme to produce CDP-choline from CTP and phosphocholine was greatly compromised. The catalytic ability of mutant enzyme D86A decreased approximately 100-fold, retaining only 1% of the wild-type kcat value with only modest changes in Km values for phosphocholine and CTP. In contrast, mutation of aspartate 115, 143, or 164 resulted in mutant enzymes that retained kcat and Km values similar to wild-type CCT. These results suggest that aspartates at position 115, 143, and 164 of rat CTP:phosphocholine cytidylyltransferase are not critical for catalysis, but may play a structural role. However, aspartate 86 may be present at or near the active site, performing an important catalytic function.