Enzymes
and Research Methodology
 Enzymes
are catalysts that are vital for living
cells to maintain their cellular activities.
The rates of reactions are increased
tremendously by enzyme catalysis.
The power of enzyme catalysis is usually
manifested through nature of the active
site where particular residues or
cofactors react with substrates, leading
to a series of chemical reactions
which require much less activation
energy when compared to an uncatalyzed
reaction.
The main goal of our research is
to gain an in-depth understanding
of the reaction mechanisms of selected
enzymes. Thorough understanding of
enzyme catalysis will lead to better
enzyme applications as catalysts for
biotechnological processes, sensors
or diagnostic tools for analytical
purposes, or drug targets for therapeutics.
To understand enzyme reaction mechanisms,
we employ various techniques and research
methods:
1. Kinetics. Kinetics allows us to
study exactly how enzymes increase
the rates of reactions. Rapid (or
pre-steady state) kinetics is used
to gain insights into each step of
catalysis. Our lab is well equipped
for studying transient kinetics: we
have two stopped-flow spectrometers
and a rapid quench apparatus. We also
perform steady-state measurements
so that data obtained from both methods
can be compared.
2. Spectroscopy. Because the enzymes
being investigated contain flavin
or PLP, which are chromogenic and
fluorescent, various types of spectroscopy
including absorbance/ fluorescence/luminescence
detection are employed to collect
thermodynamic or steady state data.
3. Kinetic isotope effects: Primary
kinetic isotope effects and solvent
kinetic isotope effects are useful
mechanistic probes to identify which
bond breakage or formation is the
rate-determining factor for the chemical
step observed in kinetic experiments.
We often investigate kinetic isotope
effects to assess whether the transfer
of group or proton of interest is
a major factor controlling catalysis.
4. Functions of active site residues.
We use site-directed mutagenesis approach
to mutate the residues speculated
to be important for the catalysis.
When data of mutants and wild-type
enzymes are compared, full understanding
of enzyme structure and function can
be developed.
5 Structures. Enzyme structures are
studied with X-ray crystallography
or NMR spectroscopy. We have been
collaborating with crystallographers
to study three-dimensional structures
of our enzymes.
Current Systems: Flavin-dependent
and PLP-dependent enzymes
Flavin-dependent and pyridoxal5-phosphate
(PLP)-dependent enzymes comprise large
numbers of enzymatic reactions that
are required for life. Flavin-dependent
enzymes play a vital role in various
biological redox reactions while PLP-dependent
enzymes are indispensable for amino
acid metabolisms. Flavins are riboflavin
derivatives that usually function
as flavoenzyme cofactors, and are
most commonly found in the forms of
flavin adenine dinucleotide (FAD)
or flavin mononucleotide (FMN). PLP
and pyridoxamine (PMP) are common
forms of vitamin B6 that function
as enzyme cofactors. Knowledge gained
from studies of these two major enzyme
classes has contributed significantly
to the understanding of cellular metabolisms
and physiology. Understanding the
reaction mechanisms of these enzymes
has resulted in many applications
in drug discovery and biotechnology.
Currently, we are conducting mechanistic
investigations of six flavin-dependent
enzymes; namely p-hydroxyphenylacetate
hydroxylase, pyranose 2-oxidase, bacterial
luciferase, 3-hydroxybenzoate 6-hydroxylase,
pyranose dehydrogenase, and alpha-glycerophosphate
oxidase, as well as a PLP-dependent
enzyme, serinehydroxymethyl transferase
from human, Plasmodium falciparum,
and Plasmodium vivax. The enzymes
used for this study are good drug
targets, useful in biotechnological
applications, or can be applied as
a gene reporter.
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