The CTP:glycerol3-phosphate cytidylyltransferase (TarD) from Staphylococcus aureus catalyzes the formation of the nucleotide activated form of glycerol 3-phosphate (CDP-glycerol) used in the construction ofteichoic acid, a structure shown to be essential in Bacillis subtilis 168. The CTP:glycerol 3-phosphate cytidylyltransferase from B. subtilis 168 (TagD) involved in teichioc acid biosynthesis has high sequence identity (69 %) and similarity (86 %) to TarD and its characterization has been well documented. In these studies, TagD was shown to carry out the CTP:glycerol 3-phosphate cytidylyltransferase reaction via a random mechanism where there is negative cooperativity in the binding of substrates but not in catalysis. The work described here illustrates that the kinetic reaction mechanism for TarD is vastly different from TagD in spite of their high sequence similarity. Recombinant TarD was over-expressed in Escherichia coli and purified to homogeneity. Steady state hi-substrate experiments were performed utilizing a high-performance liquid chromatography assay in order to deduce the kinetic mechanism for TarD. In this analysis, data were globally best fit to the model that describes the formation of a ternary complex of substrates (CTP and glycerol 3- phosphate) and enzyme before catalysis. This examination yielded Km values for CTP and glycerol 3-phosphate of 21 ± 4.1 ).lM and 36 ± 5.8 ).lM respectively, while the kcat was measured to be 2.6 ± 0.2 s -I. From the pattern observed in product inhibition studies, a classic ordered Bi Bi reaction mechanism was inferred where glycerol 3-phosphate is the initial substrate to bind followed by CTP and the release ofCDP-glycerol precedes the release of pyrophosphate. A Keq of 16 ± 15 was calculated using data obtained from exploring the kinetic parameters of the reverse reaction where data was also fit to the equation that describes the formation of a ternary complex before catalysis. The equilibrium constant was also determined experimentally to be 6. To illustrate the biological role ofTarD with respect to TagD, the integration plasmid, pSWEET, was used to introduce a copy of tarD, under xylose control, into the chromosome of a strain of B. subtilis 168 possessing a temperature sensitive mutation (tag-12) mapped to tagD. Successful complementation of the temperature sensitive mutant by tarD at the restrictive temperature indicated that despite their apparent uniqueness in kinetic mechanism, TarD and TagD have similar roles in vivo. / Thesis / Master of Science (MSc)