24 research outputs found
Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations
It has long been recognized that
the structure of a protein creates
a hierarchy of conformations interconverting on multiple time scales.
The conformational heterogeneity of the Michaelis complex is of particular
interest in the context of enzymatic catalysis in which the reactant
is usually represented by a single conformation of the enzyme/substrate
complex. Lactate dehydrogenase (LDH) catalyzes the interconversion
of pyruvate and lactate with concomitant interconversion of two forms
of the cofactor nicotinamide adenine dinucleotide (NADH and NAD<sup>+</sup>). Recent experimental results suggest that multiple substates
exist within the Michaelis complex of LDH, and they show a strong
variance in their propensity toward the on-enzyme chemical step. In
this study, microsecond-scale all-atom molecular dynamics simulations
were performed on LDH to explore the free energy landscape of the
Michaelis complex, and network analysis was used to characterize the
distribution of the conformations. Our results provide a detailed
view of the kinetic network of the Michaelis complex and the structures
of the substates at atomistic scales. They also shed light on the
complete picture of the catalytic mechanism of LDH
Conformational Heterogeneity in the Michaelis Complex of Lactate Dehydrogenase: An Analysis of Vibrational Spectroscopy Using Markov and Hidden Markov Models
Lactate
dehydrogenase (LDH) catalyzes the interconversion of pyruvate
and lactate. Recent isotope-edited IR spectroscopy suggests that conformational
heterogeneity exists within the Michaelis complex of LDH, and this
heterogeneity affects the propensity toward the on-enzyme chemical
step for each Michaelis substate. By combining molecular dynamics
simulations with Markov and hidden Markov models, we obtained a detailed
kinetic network of the substates of the Michaelis complex of LDH.
The ensemble-average electric fields exerted onto the vibrational
probe were calculated to provide a direct comparison with the vibrational
spectroscopy. Structural features of the Michaelis substates were
also analyzed on atomistic scales. Our work not only clearly demonstrates
the conformational heterogeneity in the Michaelis complex of LDH and
its coupling to the reactivities of the substates, but it also suggests
a methodology to simultaneously resolve kinetics and structures on
atomistic scales, which can be directly compared with the vibrational
spectroscopy
Enzymatic Kinetic Isotope Effects from First-Principles Path Sampling Calculations
In
this study, we develop and test a method to determine the rate
of particle transfer and kinetic isotope effects in enzymatic reactions,
specifically yeast alcohol dehydrogenase (YADH), from first-principles.
Transition path sampling (TPS) and normal mode centroid dynamics (CMD)
are used to simulate these enzymatic reactions without knowledge of
their reaction coordinates and with the inclusion of quantum effects,
such as zero-point energy and tunneling, on the transferring particle.
Though previous studies have used TPS to calculate reaction rate constants
in various model and real systems, it has not been applied to a system
as large as YADH. The calculated primary H/D kinetic isotope effect
agrees with previously reported experimental results, within experimental
error. The kinetic isotope effects calculated with this method correspond
to the kinetic isotope effect of the transfer event itself. The results
reported here show that the kinetic isotope effects calculated from
first-principles, purely for barrier passage, can be used to predict
experimental kinetic isotope effects in enzymatic systems
Targeting a Rate-Promoting Vibration with an Allosteric Mediator in Lactate Dehydrogenase
We present a new type of allosteric
modulation in which a molecule
bound outside the active site modifies the chemistry of an enzymatic
reaction through rapid protein dynamics. As a test case for this type
of allostery, we chose an enzyme with a well-characterized rate-promoting
vibration, lactate dehydrogenase; identified a suitable small molecule
for binding; and used transition path sampling to obtain ensembles
of reactive trajectories. We found that the small molecule significantly
affected the reaction by changing the position of the transition state
and, through applying committor distribution analysis, showed that
it removed the protein component from the reaction coordinate. The
ability of a small-molecule to disrupt enzymatic reactions through
alteration of subpicosecond protein motion opens the door for new
experimental studies on protein motion coupled to enzymatic reactions
and possibly the design of drugs to target these enzymes
Electric Fields and Fast Protein Dynamics in Enzymes
In recent years, there has been much
discussion regarding the origin of enzymatic catalysis and whether
including protein dynamics is necessary for understanding catalytic
enhancement. An important contribution in this debate was made with
the application of the vibrational Stark effect spectroscopy to measure
electric fields in the active site. This provided a window on electric
fields at the transition state in enzymatic reactions. We performed
computational studies on two enzymes where we have shown that fast
dynamics is part of the reaction mechanism and calculated the electric
field near the bond-breaking event. We found that the fast motions
that we had identified lead to an increase of the electric field,
thus preparing an enzymatic configuration that is electrostatically
favorable for the catalytic chemical step. We also studied the enzyme
that has been the subject of Stark spectroscopy, ketosteroid isomerase,
and found electric fields of a similar magnitude to the two previous
examples
Incorporating Fast Protein Dynamics into Enzyme Design: A Proposed Mutant Aromatic Amine Dehydrogenase
In
recent years, there has been encouraging progress in the engineering
of enzymes that are designed to catalyze reactions not accelerated
by natural enzymes. We tested the possibility of reengineering an
existing enzyme by introducing a fast protein motion that couples
to the reaction. Aromatic amine dehydrogenase is a system that has
been shown to use a fast substrate motion as part of the reaction
mechanism. We identified a mutation that preserves this fast motion
but also introduces a favorable fast motion near the active site that
did not exist in the native enzyme. Transition path sampling was used
for the analysis of the atomic details of the mechanism
Classical Molecular Dynamics Simulation of Glyonic Liquids: Structural Insights and Relation to Conductive Properties
Rhamnolipids are biosurfactants that
have obtained wide industrial
and environmental interests with their biodegradability and great
surface activity. Besides their important roles as surfactants, they
are found to function as a new type of glycolipid-based protic ionic
liquids (ILs)glyonic liquids (GLs). GLs are reported to have
impressive physicochemical properties, especially superionic conductivity,
and it was reported in experiments that specific ion selections and
the fraction of water content have a strong effect on the conductivity.
Also, the shape of the conductivity curve as a function of water fraction
in GLs is interesting with a sharp increase first and a long plateau.
We related the conductivities to the three-dimensional (3D) networks
composed of −OH inside the GLs utilizing classical molecular
dynamics (MD) simulations. The amount and size of these networks vary
with both ion species and water fractions. Before reaching the first
hydration layer, the −OH networks with higher projection/box
length ratios indicate better conductivity; after reaching the first
hydration layer and forming continuous structures, the conductivity
retains with more water molecules participating in the continuous
networks. Therefore, networks are found to be a qualitative predictor
of actual conductivity. This is explained by the analysis of the atomic
structures, including radial distribution function, fraction free
volume, anion conformations, and hydrogen bond occupancies, of GLs
and their water mixtures under different chemical conditions
Another Look at the Mechanisms of Hydride Transfer Enzymes with Quantum and Classical Transition Path Sampling
The mechanisms involved in enzymatic
hydride transfer have been
studied for years, but questions remain due, in part, to the difficulty
of probing the effects of protein motion and hydrogen tunneling. In
this study, we use transition path sampling (TPS) with normal mode
centroid molecular dynamics (CMD) to calculate the barrier to hydride
transfer in yeast alcohol dehydrogenase (YADH) and human heart lactate
dehydrogenase (LDH). Calculation of the work applied to the hydride
allowed for observation of the change in barrier height upon inclusion
of quantum dynamics. Similar calculations were performed using deuterium
as the transferring particle in order to approximate kinetic isotope
effects (KIEs). The change in barrier height in YADH is indicative
of a zero-point energy (ZPE) contribution and is evidence that catalysis
occurs via a protein compression that mediates a near-barrierless
hydride transfer. Calculation of the KIE using the difference in barrier
height between the hydride and deuteride agreed well with experimental
results
Conformational Freedom in Tight Binding Enzymatic Transition-State Analogues
Transition-state analogues of bacterial
5′-methylthioadenosine/<i>S</i>-adenosylhomocysteine
nucleosidases (MTANs) disrupt quorum-sensing
pathways in <i>Escherichia coli</i> and <i>Vibrio cholerae</i>, demonstrating the potential to limit pathogenicity without placing
bacteria under intense selective pressure that leads to antibiotic
resistance. Despite the similarity of the crystal structures of E. coli MTAN (<i>Ec</i>MTAN) and V. cholerae MTAN (<i>Vc</i>MTAN) bound
to DADMe-Immucillin-A transition-state (TS) analogues, <i>Ec</i>MTAN demonstrates femtomolar affinity for BuT-DADMe-Immucillin-A
(BDIA) whereas <i>Vc</i>MTAN possesses only picomolar affinity.
Protein dynamic interactions are therefore implicated in this inhibitor
affinity difference. We conducted molecular dynamics simulations of
both <i>Ec</i>MTAN and <i>Vc</i>MTAN in complex
with BDIA to explore differences in protein dynamic architecture.
Simulations revealed that electrostatic and hydrophobic interactions
with BDIA are similar for both enzymes and thus unlikely to account
for the difference in inhibitor affinity. The <i>Ec</i>MTAN–BDIA
complex reveals a greater flexibility and conformational freedom of
catalytically important atoms. We propose that conserved motions related
to the <i>Ec</i>MTAN transition state correlate with the
increased affinity of BDIA for <i>Ec</i>MTAN. Transition-state
analogues permitting protein motion related to formation of the transition
state are better mimics of the enzymatic transition state and can
bind more tightly than those immobilizing catalytic site dynamics
Mechanism of Cardiac Tropomyosin Transitions on Filamentous Actin As Revealed by All-Atom Steered Molecular Dynamics Simulations
The
three-state model of tropomyosin (Tm) positioning along filamentous
actin allows for Tm to act as a gate for myosin head binding with
actin. The blocked state of Tm prevents myosin binding, while the
open state allows for strong binding. Intermediate to this transition
is the closed state. The details of the transition from the blocked
to the closed state and then finally to the open state by Tm have
not been fully accessible to experiment. Utilizing steered molecular
dynamics, we investigate the work required to move the Tm strand through
the extant set of proposed transitions. We find that an azimuthal
motion around the actin filament by Tm is most probable in spite of
increased initial energy barrier from the topographical landscape
of actin