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 bacteria. This enzyme is a model for a cytidylyltransferase family that includes the enzymes that synthesize CDP-choline and CDP-ethanolamine for phosphatidylcholine and phosphatidylethanolamine biosynthesis. We have used quenching of intrinsic tryptophan fluorescence to measure binding affinities of substrates to the GCT from Bacillus subtilis. Binding of either CTP or glycerol-3-phosphate to GCT was biphasic, with two binding constants of about 0.1-0.3 and 20-40 microm for each substrate. The stoichiometry of binding was 2 molecules of substrate/enzyme dimer, so the two binding constants represented distinctly different affinities of the enzyme for the first and second molecule of each substrate. The biphasic nature of binding was observed with the wild-type GCT as well as with several mutants with altered Km or kcat values. This negative cooperativity of binding was also seen when a catalytically defective mutant was saturated with two molecules of CTP and then titrated with glycerol-3-phosphate. Despite the pronounced negative cooperativity of substrate binding, negative cooperativity of enzyme activity was not observed. These data support a mechanism in which catalysis occurs only when the enzyme is fully loaded with 2 molecules of each substrate/enzyme dimer.
Sanker
S
S
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606, USA.
Campbell
H A
HA
Kent
C
C
D007700:Kinetics; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism
D014364:Tryptophan; D009713:Nucleotidyltransferases; C020675:glycerol-3-phosphate cytidylyltransferase