Negative cooperativity of substrate binding but not enzyme activity in wild-type and mutant forms of CTP : glycerol-3-phosphate cytidylyltransferase

CTP:glycerol-3-phosphate cytidylyltransferase (GCT) catalyzes the synthesis of CDP-glycerol for teichoic acid biosynthesis in certain Gram-positive ba cteria. This enzyme is a model for a cytidylyltransferase family that inclu des the enzymes that synthesize CDP-choline and CDP-ethanolamine for phosph atidylcholine and phosphatidylethanolamine biosynthesis. We have used quenc hing of intrinsic tryptophan fluorescence to measure binding affinities of substrates to the GCT from Bacillus subtilis. Binding of either CTP or glyc erol-3-phosphate to GCT was biphasic, with two binding constants of about 0 .1-0.3 and 20-40 mum for each substrate. The stoichiometry of binding was 2 molecules of substrate/enzyme dimer, so the two binding constants represen ted distinctly different affinities of the enzyme for the first and second molecule of each substrate. The biphasic nature of binding was observed wit h the wildtype GCT as well as with several mutants with altered K-m or k(ca t) values. This negative cooperativity of binding was also seen when a cata lytically defective mutant was saturated with two molecules of CTP and then titrated with glycerol-3-phosphate. Despite the pronounced negative cooper ativity of substrate binding, negative cooperativity of enzyme activity was not observed. These data support a mechanism in which catalysis occurs onl y when the enzyme is fully loaded with 2 molecules of each substrate/enzyme dimer.