Chemistry and Biochemistry
http://hdl.handle.net/10211.3/141074
2024-03-29T06:11:45ZMonitoring proteolytic efficiency of engineered trypsin via chymotrypsinogen activity assay
http://hdl.handle.net/10211.3/214139
Monitoring proteolytic efficiency of engineered trypsin via chymotrypsinogen activity assay
Villa, Rodolfo
Protease therapeutics have been on the rise and have gained recognition for
having diverse clinical applications. One major complication faced is the abundance of
protease inhibitors that serve to regulate proteolytic activity resulting in a short therapeutic
half-life. Previous research with the model serine protease trypsin showed that residues
39 and 60 play a key role in protease: inhibitor binding. Compared to wild type, four trypsin
single variants (Y39A, Y39F, K60A, K60V) which were all catalytically similar using a
synthetic substrate displayed altered.sensitivity towards bovine pancreatic trypsin inhibitor
(BPTI) compared to wild type. In order to ascertain the viability of these engineered
variants for inhibitor resistance in vivo, the interactions between naturally occurring macro
molecular substrates were evaluated in this study. Variant Y39A displayed the highest
kcat/KM (pM'1Min-1) at 3.34 ± 0.06, while K60V displayed at 0.58 ± 0.01. Variant Y39A
activated the most chymotrypsinogen (Cg) with a total 30% activation 1.48 ± 0.05 jvM (out
of 5 pM) while K60V had the lowest with 14% or 0.70 ± 0.04 pM. With 20 nM BPTI, the
K60A variant had an activation drop of 49% while K60V dropped by 16%. At 30 nM, 50
nM and 100nM BPTI, the Y39F variant displayed greater activation than wild-type and the
K60 variants in the presence of BPTI. At higher concentrations of BPTI, the K60 variants
s have become more sensitive to inhibition, along with having lower kcat/KM and less total
chymotrypsin activation. Overall K60 variants seemed to be less proteolytically efficient
towards macromolecular substrates with less resistance towards inhibition, while Y39F
displayed promising results in both studies.
2019-01-01T00:00:00ZConjugate additions and reductive transformations of fulvenes and 6-vinylfulvenes
http://hdl.handle.net/10211.3/214111
Conjugate additions and reductive transformations of fulvenes and 6-vinylfulvenes
Ortega, Teresa de Jesus
Fulvenes are a group of cross-conjugated compounds that have been of interest due to their
diverse chemical and physical properties. Since the discovery of fulvenes by Thiele in the
1900s, and improved synthetic methods, fulvene chemistry has been explored and
applications in organometallic chemistry and natural product synthesis have been unveiled.
Herein, we disclose our results on the reactions of fulvenes and 6-vinylfulvenes with
carbon nucleophiles and reducing agents. For this study, the carbon nucleophiles selected
includes the carbon acids nitromethane and ethyl nitroacetate, furthermore, the reducing
agent consists of UAIH4. Moreover, selective diazene reduction of fulvenes has been
explored in the hopes o f gaining a convenient access to 1,2-dihydrofulvenes. Nitronate
anion additions onto regular fulvenes proved to be highly dependent on the C6-substitution
pattern of the fulvene: 6,6-disubstituted fulvenes gave spiro[2.4]hepta-4,6-dienes whereas
with 6-monosubstituted fulvenes the cyclopropanation was not completed, giving rise to 1-
and 2-substituted cyclopentadienes. Due to the improved preparative methods of 6-
vinylfulvenes, their reactivity was examined with three nucleophiles because 6-
vinylfulvenes have two electrophilic sites, at the C6 and C8 position. The nucleophilic
additions onto 6-vinylfulvenes, for the most part, occurred by a 1,4-conjugate addition with
the carbon nucleophiles and UAIH4, however, 1,2-additions were observed with a few 6-
vinylfulvene derivatives. In addition, the vinyl cyclopentadiene isomers derived from 6-
vinylfulvene with UAIH4 were employed towards the synthesis of 2-vinylsubstituted
fulvenes and isolated in moderate to excellent yields. Moreover, fulvene diazene reduction
occurred by a mono-selective endocyclic manner and 1,2-dihydrofulvenes were isolated in
moderate to excellent yields.
2019-01-01T00:00:00ZHydrothermal synthesis of pyrite phase thin films
http://hdl.handle.net/10211.3/213700
Hydrothermal synthesis of pyrite phase thin films
Mars, Diana Greenough
The pyrite phase of iron disulfide (FeS2) has attracted interest as a visible light
absorption layer for photovoltaic cells because of its low band gap and large absorption
coefficient. A one-step hydrothermal approach was employed to synthesize pyrite thin
films for solar applications. X-ray diffraction and scanning electron microscopy were
used to characterize the films and confirm the presence of the pyrite phase. Films
consisting of primarily the pyrite phase were synthesized, although crystal growth and
surface roughness were difficult to control.
2019-01-01T00:00:00ZMechanistic studies of StyA2B a self-sufficient styrene monooxygenase from R. opacus 1CP.
http://hdl.handle.net/10211.3/204022
Mechanistic studies of StyA2B a self-sufficient styrene monooxygenase from R. opacus 1CP.
Stergioulis, Alexander
Styrene Monooxygenases (SMOs) catalyze the epoxidation reaction of styrene with high
enantioselectivity, which establishes them as potential biocatalysts in chemical synthesis
and bioremediation processes. In this work, we conducted a mechanistic study on
StyA2B, a naturally fused SMO from R. opacus 1CP. The kinetic studies performed
revealed that the reductase domain of StyA2B catalyzes the reduction of FAD in a
sequential ternary mechanism. Steady-state kinetic studies of the reaction of StyA2B with
NADH and styrene showed that styrene binds weakly at low concentrations, but the
epoxidase shows strong positive cooperativity between its binding ligands. No substrate
inhibition was observed at high styrene concentrations. The addition of the partner
epoxidase, StyAl, from the StyAl/StyA2B system binds styrene non-cooperatively, but
with higher affinity and StyAl was observed to increase the catalytic activity of the
StyA2B reductase. In contrast, StyAl had no detected effect on the kinetic mechanism of
SMOB, a flavin reductase native to P. putida. This suggests that a specific protein-protein
interaction may occur in the StyAl/StyA2B system. The equilibrium midpoint potential
of FAD to StyA2B was measured to obtain information about the electronic environment
of the FAD bound to StyA2B. A significant shift of the bound-FAD midpoint potential
to a value more positive than that of free FAD was detected. This indicates that the
reduced FAD binds more tightly than oxidized FAD to StyA2B. This observation is
congruent with the steady-state ternary complex mechanism, which involves the binding
of oxidized FAD and dissociation and transport of reduced FAD from the reductase to the
epoxidase. The goal of the thesis is to provide clear description of kinetic behavior of the
StyAl/Sty A2B system. This will yield insight into unique features of this two-component
styrene monooxygenase and may help to better establish its utility as a valuable
biocatalyst.
2018-01-01T00:00:00Z