| |
| author |
Arline Tarazona
| | title |
Divalent Calcium Cation-Catalyzed Hydrolysis of Phosphodiester Bonds in Cyclic Dinucleotide Molecules
| | abstract |
The main role of the secondary messenger that is found in Vibrio cholerae bacteria, 3', 3'-
cyclic guanosine adenosine monophosphate (3', 3'-cGAMP), is to promote intestinal
colonization of the bacteria by downregulating chemotaxis through an amplified internal signal.
The cyclic molecule includes two phosphodiester bonds, known to have kinetically slow
cleavage rates, and therefore, allow these molecules to be relatively stable. Phosphodiester bonds
typically contain two negatively charged oxygen atoms, which are in resonance with each other,
that repels incoming nucleophiles that try to attack the phosphorus atom. In order to increase the
rate at which 3', 3'-cGAMP hydrolyzes and, consequently, terminate the signaling pathway,
enzymes, such as some EAL-domain or HD-GYP-domain phosphodiesterases, utilize metal-ion
cofactors to serve as catalysts. Metal-ion catalysts increase the electrophilicity of the phosphorus
and, consequently, increase the rate of hydrolysis of the secondary messenger. In enzyme active-sites,
metal-ions can also activate the nucleophile for attack on the phosphorus; however, the
mechanism by which metal-ion-catalyzed phosphodiester cleavage of 3', 3'-cGAMP occurs
under aqueous conditions is not known. After performing kinetic trials, reverse-phase high
performance liquid chromatography (HPLC) can monitor changes in substrate and reactant
concentrations, which were plotted as a function of time to produce rate constants. The
chromatographs obtained from HPLC are believed to reveal the generation of four products, 2'-AMP,
3'-AMP, 2'-GMP and 3'-GMP, with no accumulation of intermediates, which suggests
that the initial cleavage of 3', 3'-cGAMP is rate-limiting. The metal-ion-assisted hydrolysis of
3', 3'-cGAMP is validated by the breakdown of the reactant and formation of products. Plotting
rate constants as a function of Ca2+ concentrations yielded a saturating rate constant value of
1.3x10-2 min-1 and a rate constant value, at 0 M Ca2+, of 6.8x10-5 min-1, which show a catalytic
effect of 190-fold, and an equilibrium dissociation constant value of 0.48. The secondary plot
shows a rectangular hyperbola trend that is consistent with a saturating single-metal-ion
mechanism, which is also observed with RNA. Uncovering the mechanism by which 3', 3'-
cGAMP hydrolyzes can help determine whether this cleavage is consistent with the observed
RNA cleavage by the activated 2'-OH group. A second model system, 2', 3'-cyclic guanosine
adenosine monophosphate (2', 3'-cGAMP) is used to compare its products formed to the
products formed of 3', 3'-cGAMP in Ca2+ solution. Comparing between the 3', 3'-cGAMP and
2', 3'-cGAMP reactions can reveal how slight changes in phosphate linkages can impact the
2'OH- induced intramolecular attack on the electrophilic phosphorus. One of the effects as a
result of the changes in phosphate linkages, for instance, can be seen in the catalytic defect of
about 30-times in the rate constant for the hydrolysis reaction, when the substrate is changed
from 2', 3'-cGAMP to 3', 3'-cGAMP.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.A. (2019)
|
| advisor |
Adam Cassano
|
| committee |
Kimberly Choquette
Lisa Jordon
|
| full text | AMTarazona.pdf |
| |
