THE ASSOCIATION OF LIPID ACTIVATORS WITH THE AMPHIPATHIC HELICAL DOMAIN OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE ACCELERATES CATALYSIS BY INCREASING THE AFFINITY OF THE ENZYME FOR CTP

The biochemical mechanism for the regulation of enzyme activity by lipid modulators and the role of the amphipathic alpha-helical domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated by analyzing the kinetic properties of the wild-type protein and two truncation mutants isolated from a baculovirus expression system. The CT[Delta 312-367] mutant protein lacked the carboxyl-terminal phosphorylation domain and retained high catalytic activity along with both positive and negative regulation by lipid modulators. The CT[Delta 257-367] deletion removed in addition the region containing three consecutive amphipathic alpha-helical repeats The CT[Delta 257-367] mutant protein exhibited a significantly lower specific activity compared to CT or CT[Delta 312-367] when expressed in either insect or mammalian cells; however, CT[Delta 257-367] activity was refractory to either stimulation or inhibition by lipid regulators. Lipid activators accelerated CT activity by decreasing the K-m for CTP from 24.7 mM in their absence to 0.7 mM in their presence. The K-m for phosphocholine was not affected by lipid activators. The activity of CT[Delta 257-367] was comparable to the activity of wildtype CT in the absence of lipid activators and the CTP K-m for CT[Delta 257-367] was 13.9 mM. The enzymatic properties of the CT[Delta 231-367] mutant were comparable to those exhibited by the CT[257-367] mutant indicating that removal of residues 231 through 257 did not have any additional influence on the lipid regulation of the enzyme. Thus, the region between residues 257 and 312 was required to confer lipid regulation on CT, and the association of activating lipids with this region of the protein stimulated catalysis by increasing the affinity of the enzyme for CTP.