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)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22714 |
| Date | 03 1900 |
| Creators | Badurina, David |
| Contributors | Brown, Eric D., Biochemistry |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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