Absolute Configuration of Bromochlorofluoromethane from Molecular Dynamics Simulation of its Enantioselective Complexation by Cryptophane-CJ. Costante-Crassous, T.J. Marrone, J.M. Briggs, J.A. McCammon and A. ColletJournal of the American Chemical Society, Vol. 119, pp. 3818-3823 (1997)
Earlier NMR experiments have shown that the inclusion of
bromochlorofluoromethane (CHFClBr) 1 within the cavity of
(-)-cryptophane-C 2 in chloroform solution is enantioselective and that
(-)-1 is more strongly bound than (+)-1 with a free energy difference
(delta delta G) of 1.1 kJ/mol. In order to gain information on the
relative configuration of the diastereomeric complexes and hence on the
absolute configuration of 1, we have tried to reproduce these
experiments by computational methods, and we have calculated the free
energy difference for the binding of (R) and (S)-1 to (-)-2. For this
purpose, the OPLS parameters for CHFClBr were optimized by Monte Carlo
(MC) simulations of the pure liquid. Then molecular dynamics (MD)
simulations were performed on the host-guest system in a solvent box of
chloroform using multiconfiguration thermodynamic integration (MCTI) and
free energy perturbation (FEP) methods to calculate the free energy
difference between the diastereomeric complexes. The (R)-1@(-)-2 complex
was thus calculated to be more stable than the (S)-1@(-)-2 one by 0-2.6
kJ/mol, which is of the same order of magnitude as the experimental
result. Since the (-)-1@(-)-2 complex is more stable than the
(+)-1@(-)-2 one, and since the absolute configuration of 2 is known, it
was concluded that the absolute configuration of CHFClBr must be (R)-(-)
(or (S)-(+)); this conclusion is in agreement with a recent independent
assignment based on Raman Optical Activity studies.
Simulation of Electrostatic and Hydrodynamic Properties of Serratia EndonucleaseJan Antosiewicz, Mitchell D. Miller, Kurt L. Krause and J. Andrew McCammonBiopolymers, Vol. 41, Issue 4, pp. 443-450 (1997) [PubMed 9080779]We analyze the electrostatic and hydrodynamic properties of a nuclease
from the pathogenic Gram-negative bacterium Serratia marcescens using
finite-difference Poisson-Boltzmann methods for electrostatic
calculations and a bead-model approach for diffusion coefficient
calculations. Electrostatic properties are analyzed for the enzyme in
monomeric and dimeric forms and also in the context of DNA binding by
the nuclease. Our preliminary results show that binding of a dsDNA
dodecamer by nuclease, causes an overall shift in the charge of the
protein by approximately three units of elementary charge per monomer
resulting in a positively charged protein at physiologic pH. In these
calculations, the free enzyme was found to have a negative (-1e) charge
per monomer at pH7. The most dramatic shift in pKa involves His 89 whose
pKa increases by three pH units upon DNA binding. This shift leads to a
protonated residue at pH7, in contrast to the unprotonated form in the
free enzyme. DNA binding also leads to a decrease in the energetic
distances between the most stable protonation states of the enzyme.
Dimerization has no significant effect on the electrostatic properties
of each of the monomers for both free enzyme and that bound to DNA.
Results of hydrodynamic calculations are consistent with the dimeric
form of the enzyme in solution. The computed translational diffusion
coefficient for the dimer model of the enzyme is in very good agreement
with measurements from light scattering experiments. Preliminary
electro-optical calculations indicate that the dimer should possess a
large dipole moment (approximately 600 Debye units) as well as
substantial optical anisotropy (limiting reduced linear electric
dichroism of about 0.3). Therefore, this system may serve as a good
model for investigation of electric and hydrodynamic properties by
relaxation electro-optical experiments.
Structure-Based Drug Design: Computational AdvancesT.J. Marrone, J.M. Briggs and J.A. McCammonAnnual Review of Pharmacology and Toxicology, Vol. 37, No. 1, pp. 71-90 (1997) [PubMed 9131247]Structure-based computational methods continue to show progress in the
discovery and refinement of therapeutic agents. Several such methods and
their applications are described. These include molecular modeling,
docking, fragment methods, 3D database techniques, and free energy
perturbation. Related issues that are discussed include the use of
simplified energy functions and the determination of the positions of
tightly-bound waters. Strengths and weaknesses of the various methods
are described.
Application of Poisson-Boltzmann Solvation Forces to Macromolecular SimulationsA.H. Elcock, M.J. Potter and J.A. McCammonIn "Computer Simulation of Biomolecular Systems," Vol. 3, W.F. van Gunsteren, P.K. Weiner, A.J. Wilkinson, Eds., Kluwer Academic Publishers, Dordrecht, pp. 244-261 (1997)
One of the most difficult problems encountered in the dynamical
simulation of large macromolecular systems is how to deal adequately
with the huge number of atomic interactions involved. For aqueous phase
simulations the computational burden associated with solvent water
molecules can easily outstrip that associated with the macromolecule,
even though the behavior of the solvent itself may not be of much
interest. Not surprisingly therefore considerable interest has been
focused on the use of methods in which explicit solvent water molecules
are replaced by an implicit dielectric continuum representation. Perhaps
the most generally accepted continuum-based approach centers on the use
of the Poisson-Boltzmann (PB) equation of classical electrostatics, a
method which owes its success to the fact that many solvation-related
phenomena (with the notable exception of the hydrophobic effect) appear
to be essentially electrostatic in nature. Until very recently, use of
the PB approach has largely been restricted to calculations involving
static representations of molecular structure, but the recent
development of methods to obtain solvation forces from the PB equation
means that it can now in principle also be used in dynamics simulations.
Applications of the former type have been comprehensively reviewed in
the literature and are not discussed further in this article; instead,
we restrict our attention here to the potential use of PB electrostatics
in dynamical simulations of macromolecules.
Enzyme-Inhibitor Association Thermodynamics: Explicit and Continuum Solvent StudiesHaluk Resat, Tami J. Marrone and J. Andrew McCammonBiophysical Journal, Vol. 72, No. 2, pp. 522-532 (1997) [PubMed 9017183]Studying the thermodynamics of biochemical association reactions at the
microscopic level requires efficient sampling of the configurations of
the reactants and solvent as a function of the reaction pathways. In
most cases, the associating ligand and receptor have complementary
interlocking shapes. Upon association, loosely connected or disconnected
solvent cavities at and around the binding site are formed. Disconnected
solvent regions lead to severe statistical sampling problems when
simulations are performed with explicit solvent. It was recently
proposed that, when such limitations are encountered, they might be
overcome by use of the grand canonical ensemble
http://dx.doi.org/10.1021/jp951496n" target="_blank" class="ref">Resat
et al., J. Phys. Chem. 100, 1426 (1996). Here, we investigate one such
case and report the association free energy profile (potential of mean
force) between trypsin and benzamidine along a chosen reaction
coordinate as calculated using the grand canonical Monte Carlo method.
The free energy profile is also calculated for a continuum solvent model
using the Poisson equation, and the results are compared to the explicit
water simulations. The comparison shows that the continuum solvent
approach is surprisingly successful in reproducing the explicit solvent
simulation results. The Monte Carlo results are analyzed in detail with
respect to solvation structure. In the binding site channel, there are
waters bridging the carbonyl oxygen groups of Asp189 with the NH(2)
groups of benzamidine which are displaced upon inhibitor binding. A
similar solvent-bridging configuration has been seen in the crystal
structure of trypsin complexed with bovine pancreatic trypsin inhibitor.
The predicted locations of other internal waters are in very good
agreement with the positions found in the crystal structures which
supports the accuracy of the simulations.
Electrostatic effects in homeodomain-DNA interactionsF. Fogolari, A.H. Elcock, G. Esposito, P. Viglino, J.M. Briggs and J.A. McCammonJournal of Molecular Biology, Vol. 267, pp. 368-381 (1997) [PubMed 9096232]Electrostatics plays an important role in protein-DNA association. The
Poisson-Boltzmann equation is a powerful tool to study these effects. It
allows free energies of association to be calculated and allows detailed
analyses of the electrostatic dependence of thermodynamic quantities to
be made. We report here an investigation on the role of electrostatics
in homeodomain-DNA interaction. Compared to the proteins considered in
previous investigations of protein-DNA systems, the homeodomain is a
highly charged small protein and forms extensive ion pairs upon binding
DNA. It is not obvious, therefore, whether the previous results extend
to this class of molecules. We investigated the salt dependence of the
binding constant for specific association and for different models of
non-specific association. Our results indicate that counterion release
accounts for a significant fraction of the salt dependence of the free
energy. Thermodynamic data for some specific homeodomain mutants can be
explained by favorable electrostatic interactions in the major groove of
DNA.
Prediction of Titration Properties of Structures of a Protein Derived from Molecular Dynamics TrajectoriesS.T. Wlodek, J. Antosiewicz and J.A. McCammonProtein Science, Vol. 6, Issue 2, pp. 373-382 (1997) [PubMed 9041639]This paper explores the dependence of the molecular dynamics (MD)
trajectory of a protein molecule upon the titration state assigned to
the molecule. Four 100 ps molecular dynamics trajectories of bovine
pancreatic trypsin inhibitor (BPTI) were generated, starting from two
different structures, each of which was held in two different charge
states. The two starting structures were the X-ray crystal structure and
one of the solution structures determined by NMR, and the charge states
differed only in the ionization state of N terminus. Although it is
evident that the MD simulations were too short to fully sample the
equilibrium distribution of structures in each case, standard
Poisson-Boltzmann titration state analysis of the resulting
configurations shows general agreement between the overall titration
behavior of the protein and the charge state assumed during MD
simulation: at pH 7, the total net charge of the protein resulting from
the titration analysis is consistently lower for the protein with the N
terminus assumed to be neutral than for the protein with the N terminus
assumed to be charged. For most of the ionizable residues, the
differences in the calculated pKa's among the four trajectories are
statistically negligible, and remain in good agreement with the data
obtained by crystal structure titration and by experiment. The
exceptions include the N terminus which directly responds to the change
of its imposed charges, the C terminus which in the NMR structure
strongly interacts with the former, and a few other residues, (Arg1,
Glu7, Tyr35 and Arg42) whose pKa's reflect the initial structure and the
limited trajectory lengths. This study illustrates the importance of the
careful assignment of protonation states at the start of MD simulations,
and points to the need for simulation methods that allow for the
variation of the protonation state in the calculation of equilibrium
properties.
The Statistical-Thermodynamic Basis for Computation of Binding Affinities: A Critical ReviewM.K. Gilson, J.A. Given, B.L. Bush and J.A. McCammonBiophysical Journal, Vol. 72, No. 3, pp. 1047-1069 (1997) [PubMed 9138555]The noncovalent association of molecules is of central importance in
biology and pharmacology. It underlies the action of hormones, the
control of DNA transcription, the recognition of antigens by the immune
system, the catalysis of chemical reactions by enzymes, and the actions
of many drugs. Therefore, methods of predicting the affinity of such
noncovalent association would be of great practical value. For example,
predictive computational models for the noncovalent association of
biomolecules and their ligands would be useful in structure-based drug
design and in the redesign of enzymes. As recently reviewed, many groups
have invested ingenuity and effort into the development of such models.
However, although the physical chemistry of these association processes
is rooted in statistical themodynamics, few models are explicitly
derived from these underlying principles. This has led to a certain
amount of confusion in the field, as discussed below. The present paper
therefore presents a statistical-thermodynamic derivation of the
standard free energy of binding, and uses this to review the
underpinnings of methods for computing noncovalent binding affinities.
Rational Design of HIV Protease InhibitorsC.N. Hodge, T.P. Straatsma, J.A. McCammon and A. WlodawerIn "Structural Biology of Viruses," W. Chiu, R.M. Burnett, and R. Garcea, Eds., Oxford University Press, pp. 451-473 (1997)
Replication of the human immunodeficiency virus (HIV) requires the
action of a virally encoded protease to produce components of progeny
virions within infected cells. Therefore much effort has been devoted to
developing specific inhibitors of protease in the hope that these may
prove valuable in the management of HIV infections. Fortunately, the
structures of protease and many of its inhibitor complexes have been
determined by X-ray crystallography. This chapter describes how such
structural data are being used in computer modeling and computer
simulation studies to speed the discovery of clinically useful protease
inhibitors. The first section outlines the crystallographic results that
have been obtained for protease-inhibitor complexes. Subsequent sections
describe how computer modeling has helped in the discovery of a
promising new class of protease inhibitors, and how free energy
simulations can help in deciding which inhibitors within a given class
will have the highest affinity for protease.
Weighted-Ensemble Simulated Annealing: Faster Optimization on Hierarchical Energy SurfacesG.A. Huber and J.A. McCammonPhysical Review E, Vol. 55, pp. 4822-4825 (1997)
A new method, Weighted-Ensemble Annealing, is proposed for finding the
global minima of complicated functions, such as those found in
biological problems. This method performs simulated annealing using
multiple system copies; it automatically adjusts the distribution of
copies and the allocation of computer resources as the cooling proceeds.
This readjustment procedure is designed to take advantage of the
hierarchical structure of the energy landscape of biomolecules and other
systems. This method is applied to a fractal-like function with energy
barriers of many sizes and a large entropy barrier. It is shown that
using an optimnal number of system copies results in a success rate for
finding the global minimum which is an order of magnitude higher that
the success rate from traditional single-copy annealing, using the same
total number of function evaluations.
Conformational Sampling with Poisson-Boltzmann Forces and a Stochastic Dynamics/Monte Carlo Method: Application to Alanine DipeptideJ.L. Smart, T.J. Marrone and J.A. McCammonJournal of Computational Chemistry, Vol. 18, pp. 1750-1759 (1997)
We apply a combination of stochastic dynamics and Monte Carlo methods
(MC/SD) to the alanine dipeptide, with solvation forces derived from a
Poisson- Boltzmann model supplemented with apolar terms. Our purpose is
to study the effects of the model parameters, such as the friction
constant and the size of the electrostatic finite difference grid, on
the rate of conformational sampling and on the accuracy of the resulting
free energy map. For dialanine, a converged Ramachandran map is produced
in significantly less time than what is required by stochastic dynamics
or Monte Carlo alone. MC/SD is also shown to be faster, per timestep,
than explicit methods.
Modeling the Electrophoresis of Lysozyme II. Inclusion of Ion RelaxationS.A. Allison, M. Potter and J.A. McCammonBiophysical Journal, Vol. 73, No. 1, pp. 133-140 (1997) [PubMed 9199778]In this work, boundary element methods are used to model the
electrophoretic mobility of lysozyme over the pH range 2-6. The model
treats the protein as a rigid body of arbitrary shape and charge
distribution derived from the crystal structure. Extending earlier
studies, the present work treats the equilibrium electrostatic potential
at the level of the full Poisson-Boltzmann (PB) equation and accounts
for ion relaxation. This is achieved by solving simultaneously the
Poisson, ion transport, and Navier-Stokes equations by an iterative
boundary element procedure. Treating the equilibrium electrostatics at
the level of the full rather than the linear PB equation, but leaving
relaxation out, does improve agreement between experimental and
simulated mobilities. Including ion relaxation improves it even more.
The effects of nonlinear electrostatics and ion relaxation are greatest
at low pH where the net charge on lysozyme is greatest. In the absence
of relaxation, a linear dependence of mobility and average polyion
surface potential is observed.
Colloquium on Signaling and Molecular Structure in PharmacologyP. Taylor, E.M. Ross, P.C. Sternweis, R. Neubig and J.A. McCammonMolecular Pharmacology, Vol. 52, pp. 1-5 (1997) [PubMed 9224805]The first international meeting of major biological research societies
within a supercomputer center is described. The meeting occurred in
March 1997 at the San Diego Supercomputer Center. The resources of the
center allowed the audience to view biomolecular structure and dynamics
in three dimensions as the speakers delivered their lectures.
Structural Fluctuations of a Cryptophane-Tetramethylammonium Host-Guest System: A Molecular Dynamics SimulationP.D. Kirchhoff, J.-P. Dutasta, A. Collet and J.A. McCammonJournal of the American Chemical Society, Vol. 119, pp. 8015-8022 (1997)
Cryptophanes are aromatic hosts which bind a variety of guests. Here, we
describe a 25 nanosecond molecular dynamics simulation of a particular
cryptophane host-guest complex in water. The cryptophane used in this
study was used previously in a 20 nanosecond molecular dynamics
simulation to describe the fluctuations of the uncomplexed host. This
cryptophane features three pores which open onto a cavity where the
guests bind. In the current study, tetramethylammonium ion (TMA+) has
been placed within the cryptophane cavity to form the host-guest
complex. The molecular dynamics simulation in combination with a
surfacing algorithm provides information on the frequency with which the
cryptophane pores open wide enough to admit or release guest molecules
of any given size. We discuss these fluctuations and their possible
consequences for binding kinetics, making comparisons between the
cryptophane and the cryptophane-TMA+ complex.
Electrostatic Influence on the Kinetics of Ligand Binding to Acetylcholinesterase: Distinctions Between Active Center Ligands and FasciculinZoran Radi&Journal of Biological Chemistry, Vol. 272, No. 37, pp. 23265-23277 (1997) [PubMed 9287336]To explore the role that surface and active center charges play in
electrostatic attraction of ligands to the active center gorge of
acetylcholinesterase (AChE), and the influence of charge on the reactive
orientation of the ligand, we have studied the kinetics of association
of cationic and neutral ligands with the active center and peripheral
site of AChE. Electrostatic influences were reduced by sequential
mutations of six surface anionic residues outside of the active center
gorge (Glu84, Glu91, Asp280, Asp283, Glu292 and Asp372), and three
residues within the active center gorge (Asp74 at the rim, Glu202 and
Glu450 at the base). The peripheral site ligand, fasciculin2 (FAS2), a
peptide of 6.5 kD with a net charge of +4, shows a marked enhancement of
rate of association with reduction in ionic strength, and this ionic
strength dependence can be markedly reduced by progressive
neutralization of surface and active center gorge anionic residues. By
contrast, neutralization of surface residues only has a modest influence
on the rate of cationic m-trimethylammonio trifluoroacetophenone (TFK+)
association with the active serine, whereas neutralization of residues
in the active center gorge has a marked influence on the rate but with
little change in the ionic strength dependence. Brownian dynamics
calculations for approach of a small cationic ligand to the entrance of
the gorge show the influence of individual charges to be in quantitative
accord with that found for the surface residues. Anionic residues in the
gorge may help to orient the ligand for reaction or to trap the ligand.
Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the
active center, but inverts the ionic strength dependence for the
cationic TFK' association with AChE. Hence it appears that TFK' must
traverse an electrostatic barrier at the gorge entry imparted by the
bound FAS2 with its net charge of +4.
Molecular Properties of Amphotericin B Membrane Channel - A Molecular Dynamics SimulationM. Baginski, H. Resat and J.A. McCammonMolecular Pharmacology, Vol. 52, pp. 560-570 (1997) [PubMed 9380018]Amphotericin B is a powerful, but also toxic, anti-fungal antibiotic
used to treat systemic infections. It forms ionic membrane channels in
fungal cells. These antibiotic-sterol channels are responsible for the
leakage of ions which causes cell destruction. The detailed molecular
properties and structure of amphotericin B channels are still unknown.
In the present study, two molecular dynamic simulations of a particular
model AmB-cholesterol channel were performed. The water and phospholipid
environment were included in our simulations, and the results obtained
were compared with available experimental data. It was found that it is
mainly the hydrogen bonding interactions which keep the channel stable
in its open form. Our study also revealed the important role of the
intermolecular interactions between the hydroxyl, amino and carboxyl
groups of the channel forming molecules. Particularly, some hydroxyl
groups stand out as new "hot spots" potentially useful for
chemotherapeutic investigations. Our results also help to understand why
certain antibiotic derivatives, with a blocked amino group, are less
active. We also present a hypothesis for the role of membrane lipids and
cholesterol in the channel.
Molecular Dynamics of Acetylcholinesterase Dimer Complexed with TacrineS.T. Wlodek, T.W. Clark, L.R. Scott and J.A. McCammonJournal of the American Chemical Society, Vol. 119, pp. 9513-9522 (1997)
We have studied the dynamic properties of acetylcholinesterase dimer
from Torpedo californica liganded with tacrine (AChE-THA) in solution
using molecular dynamics. The simulation reveals fluctuations in the
width of the primary channel to the active site that are large enough to
admit substrates. Alternative entries to the active site through the
side walls of the gorge have been detected in a number of structures.
This suggests that transport of solvent molecules participating in
catalysis can occur across the porous wall, contributing to the
efficiency of the enzyme.
Kinase Conformations: A Computational Study of the Effect of Ligand BindingVolkhard Helms and J. Andrew McCammonProtein Science, Vol. 6, Issue 11, pp. 2336-2343 (1997) [PubMed 9385635]Protein function is often controlled by ligand-induced conformational
transitions. Yet, in spite of the increasing number of three-dimensional
crystal structures of proteins in different conformations, not much is
known about the driving forces of these transitions. As an initial step
towards exploring the conformational and energetic landscape of protein
kinases by computational methods, intramolecular energies and hydration
free energies were calculated for different conformations of the
catalytic domain of cAMP-dependent protein kinase (cAPK) with a
continuum (Poisson) model for the electrostatics. Three protein kinase
crystal structures for ternary complexes of cAPK with the peptide
inhibitor PKI(5-24) and ATP or AMP-PNP were modelled into idealized
intermediate and open conformations. Concordant with experimental
observation, we find that the binding of PKI(5-24) is more effective in
stabilizing the closed and intermediate forms of cAPK than ATP.
PKI(5-24) seems to drive the final closure of the active site cleft from
intermediate to closed state since ATP does not distinguish between
these two states. Binding of PKI(5-24) and ATP is energetically additive.
A Continuum Solvation Model for Studying Protein Hydration Thermodynamics at High TemperaturesA.H. Elcock and J.A. McCammonJournal of Physical Chemistry B, Vol. 101, pp. 9624-9634 (1997)
A macroscopic solvation model which combines a solvent-accessible
surface area term to describe hydration of non-polar groups with a
continuum electrostatics term to describe the hydration of polar groups
has previously been shown to provide an excellent description of amino
acid hydration free energies at 25 degrees centigrade
(http://dx.doi.org/10.1021/j100058a043" target="_blank"
class="ref">Sitkoff et al. 1994, J. Phys. Chem. vol. 98. pp. 1978-1988).
We describe here the extension of this method and its accompanying
parameter set (known as PARSE) to handle temperatures in the range from
5 degrees to 100 degrees centigrade. For the neutral amino acids,
hydration free energies were taken from the literature; for the charged
amino acids Asp, Glu, and Lys, hydration free energies were obtained by
combining results for the neutral analogues with information on the pKa
value at the required temperature. An important result of this analysis
is that the hydration free energies of the charged residues are much
more strongly affected by increasing temperature than their neutral
analogues. In extending the PARSE method to reproduce hydration free
energies over a range of temperatures, a number of alternative models
were investigated: best results were obtained when separate surface area
dependent terms were used to represent the hydration of aliphatic and
aromatic regions and when the continuum electrostatics term was made
strongly temperature dependent. The temperature dependence of the
electrostatic component stems partly from changes in the dielectric
constant of water, but appears to be rather better described when the
atomic radii are also made temperature dependent, increasing in size as
the temperature rises. This requirement for temperature dependent radii
gains important support from previous studies using the Born model to
descibe the entropies of hydration of simple ions. The extension of the
PARSE method described here permits its use in investigating the effects
of hydration on protein stability over a wide range of
biologically-relevant temperatures.
pH Dependence of Antibody/Lysozyme ComplexationC.J. Gibas, S. Subramaniam, J.A. McCammon, B.C. Braden and R.J. PoljakBiochemistry, Vol. 36, No. 50, pp. 15599-15614 (1997) [PubMed 9398288]Association between proteins often depends on the pH and ionic strength
conditions of the medium in which it takes place. This is especially
true in complexation involving titratable residues at the complex
interface. Continuum electrostatics methods were used to calculate the
pH-dependent energetics of association of hen-egg lysozyme with two
closely related monoclonal antibodies raised against it and the
association of these antibodies against an avian species variant. A
detailed analysis of the energetic contributions reveals that even
though the hallmark of association in the two complexes is the presence
of conserved charged-residue interactions, the environment of these
interactions significantly influences the titration behavior and
concomitantly the energetics. The contributing factors include, minor
structural rearrangements, buried interfacial area, dielectric
environment of the key titratable residues and the geometry of the
residue dispositions. Modeled structures of several mutant complexes
were also studied so as to further delineate the contribution of
individual factors to the titration behavior.
Electrostatic Channeling of Substrates Between Enzyme Active Sites: Comparison of Simulation and ExperimentA.H. Elcock, G.A. Huber and J.A. McCammonBiochemistry, Vol. 36, No. 51, pp. 16049-16058 (1997) [PubMed 9405038]Recent simulation work has indicated that channeling of charged
substrates between the active sites of bifunctional enzymes or bienzyme
complexes can be significantly enhanced by favorable interactions with
the electrostatic field of the enzymes. The results of such simulations
are expressed in terms of transfer efficiencies, which describe the
probability that substrate leaving the active site of the first enzyme
will reach the active site of the second enzyme before escaping out into
bulk solution. The experimental indicators of channeling on the other
hand, are factors such as a decrease in the transient (lag) time for
appearance of the final product of the coupled enzyme reaction, or a
decreased susceptibility of the overall reaction rate to the presence of
competing enzymes or competitive inhibitors. The work reported here aims
to establish a connection between the transfer efficiencies obtained
from simulation, with the above-mentioned experimental observables. We
accomplish this by extending previously reported analytical approaches
to combine the simulated transfer efficiency with the Michaelis-Menten
kinetic parameters K
m and V
max of the enzymes
involved; expressions are derived to allow both transient times and
steady state rates to be calculated. We then apply these results to the
two systems which have been studied both theoretically and
experimentally. In the first case, that of the bifunctional enzyme
dihydrofolate reductase-thymidylate synthase (DHFR-TS), the
experimentally observed decrease in transient times is found to be
consistent with a transfer efficiency of greater than or equal to 80%.
In the second case, that of a citrate synthase-malate dehydrogenase
(CS-MDH) fusion protein, a transfer efficiency of 73% is consistent with
the experimental transient time measurements. Separate and independent
analysis of the effects of adding the competing enzyme aspartate
aminotransferase (AAT) gives a transfer efficiency of 69%, in excellent
agreement with the transient time results. The transfer efficiencies
thus obtained from experimental results are in both cases in reasonable
agreement with those obtained from simulation. One important discrepancy
between simulation and experiment is however found in the reported
effects of adding a competitive inhibitor in the DHFR-TS system:
qualitatively different results are expected from the theoretical
analysis. A possible reason for this apparent contradiction is discussed.
Quantum Classical Molecular Dynamics Simulation of Phospholipid HydrolysisP. Ba&HyperNews, Vol. 2, pp. 18-21 (1997)
Recent advances in the use of quantum dynamical simulations for the
study of enzymatic catalytis are described.