interacts with the TS domain and aids in stabilizing the interdomain
attachment. The junction region contains the a j1 helix which links
the DHFR and TS domains and interacts with the DHFR domain
of the other polypeptide chain [42]. These features and the major
differences in amino acid sequence, structure, and function
between Plasmodial DHFR-TS and bacterial and vertebrate
DHFRs have allowed for the development of species-specific
inhibitors.
3 Ligand-Dependent Conformational Dynamics during DHFR Catalysis
The first indication that conformational dynamics may play a role in
ecDHFR catalysis came from kinetic measurements, which showed
that the apoenzyme exists as two isoforms, E1 and E2 [43–45]. In
these experiments, the authors made stopped-flow measurements
of association and dissociation rates of ligands to and from the
enzyme. They mixed together solutions of the enzyme and ligand
and recorded the resulting tryptophan fluorescence over time using
the excitation wavelength of 290 nm and a 341 nm interference
filter. Alternatively, the authors excited the tryptophans and
recorded the enhancement of coenzyme (NADPH) fluorescence
by energy transfer using an excitation wavelength of 290 nm and a
449 nm interference filter. The data was analyzed to obtain the rates
for formation and dissociation of binary complexes of both forms
of the enzyme. The second isoform was detected as a slow ligandindependent phase that followed an initial ligand-dependent burst
phase when mixed with NADPH and substrate or inhibitor in a
stopped-flow fluorescence experiment by Dunn et al. [43–45] They
found that NADPH appears to bind rapidly and exclusively to the
E1 isoform and that binding of a ligand or inhibitor to either the
substrate or cofactor sites resolves the ambiguity between conformational states and allows a single conformation that readily binds
further ligands to form ternary complexes to be observed
[18]. This observation highlights the connection between protein
conformation and ligand binding.
In a more recent study, Reddish et al. used tryptophan fluorescence probed temperature-jump spectroscopy to observe the kinetics of ligand binding and ligand-induced conformational changes of
three DHFR complexes to attempt to establish the relationship
between conformational changes and catalytic steps along the
DHFR pathway [46]. Temperature-jump spectroscopy can be
used to measure rapid reaction rates in the microsecond timescale,
which is a significant timescale for catalysis and allostery. In this
method, temperature is rapidly increased perturbing the system.
The fluorescence of the system is then observed as the system
reaches equilibrium with a new equilibrium constant. The three
196
Melanie Goldstein and Nina M. Goodey
attachment. The junction region contains the a j1 helix which links
the DHFR and TS domains and interacts with the DHFR domain
of the other polypeptide chain [42]. These features and the major
differences in amino acid sequence, structure, and function
between Plasmodial DHFR-TS and bacterial and vertebrate
DHFRs have allowed for the development of species-specific
inhibitors.
3 Ligand-Dependent Conformational Dynamics during DHFR Catalysis
The first indication that conformational dynamics may play a role in
ecDHFR catalysis came from kinetic measurements, which showed
that the apoenzyme exists as two isoforms, E1 and E2 [43–45]. In
these experiments, the authors made stopped-flow measurements
of association and dissociation rates of ligands to and from the
enzyme. They mixed together solutions of the enzyme and ligand
and recorded the resulting tryptophan fluorescence over time using
the excitation wavelength of 290 nm and a 341 nm interference
filter. Alternatively, the authors excited the tryptophans and
recorded the enhancement of coenzyme (NADPH) fluorescence
by energy transfer using an excitation wavelength of 290 nm and a
449 nm interference filter. The data was analyzed to obtain the rates
for formation and dissociation of binary complexes of both forms
of the enzyme. The second isoform was detected as a slow ligandindependent phase that followed an initial ligand-dependent burst
phase when mixed with NADPH and substrate or inhibitor in a
stopped-flow fluorescence experiment by Dunn et al. [43–45] They
found that NADPH appears to bind rapidly and exclusively to the
E1 isoform and that binding of a ligand or inhibitor to either the
substrate or cofactor sites resolves the ambiguity between conformational states and allows a single conformation that readily binds
further ligands to form ternary complexes to be observed
[18]. This observation highlights the connection between protein
conformation and ligand binding.
In a more recent study, Reddish et al. used tryptophan fluorescence probed temperature-jump spectroscopy to observe the kinetics of ligand binding and ligand-induced conformational changes of
three DHFR complexes to attempt to establish the relationship
between conformational changes and catalytic steps along the
DHFR pathway [46]. Temperature-jump spectroscopy can be
used to measure rapid reaction rates in the microsecond timescale,
which is a significant timescale for catalysis and allostery. In this
method, temperature is rapidly increased perturbing the system.
The fluorescence of the system is then observed as the system
reaches equilibrium with a new equilibrium constant. The three
196
Melanie Goldstein and Nina M. Goodey