Ram Prasad Bora

Perspective: Defining and quantifying the role of dynamics in enzyme catalysis

Enzymes control chemical reactions that are key to life processes, and allow them to take place on the time scale needed for synchronization between the relevant reaction cycles. In addition to general interest in their biological roles, these proteins present a fundamental scientific puzzle, since the origin of their tremendous catalytic power is still unclear. While many different hypotheses have been put forward to rationalize this, one of the proposals that has become particularly popular in recent years is the idea that dynamical effects contribute to catalysis. Here, we present a critical review of the dynamical idea, considering all reasonable definitions of what does and does not qualify as a dynamical effect. We demonstrate that no dynamical effect (according to these definitions) has ever been experimentally shown to contribute to catalysis. Furthermore, the existence of non-negligible dynamical contributions to catalysis is not supported by consistent theoretical studies. Our review is aimed, in part, at readers with a background in chemical physics and biophysics, and illustrates that despite a substantial body of experimental effort, there has not yet been any study that consistently established a connection between an enzymes conformational dynamics and a significant increase in the catalytic contribution of the chemical step. We also make the point that the dynamical proposal is not a semantic issue but a well-defined scientific hypothesis with well-defined conclusions.

Dimerization of the full-length Alzheimer amyloid β-peptide (Aβ42) in explicit aqueous solution: a molecular dynamics study.

In this study, the mechanism of dimerization of the full-length Alzheimer amyloid beta (Aβ42) peptide and structural properties of the three most stable dimers have been elucidated through 0.8 μs classical molecular dynamics (MD) simulations. The Aβ42 dimer has been reported to be the smallest neurotoxic species that adversely affects both memory and synaptic plasticity. On the basis of interactions between the distinct regions of the Aβ42 monomer, 10 different starting configurations were developed from their native folded structures. However, only six of them were found to form dimers and among them the three most stable (X(P), C-C(AP), and N-N(P)) were chosen for the detailed analysis. The structural properties of these dimers were compared with the available experimental and theoretical data. The MD simulations show that hydrophobic regions of both monomers play critical roles in the dimerization process. The high content of the α-helical structure in all the dimers is in line with its experimentally proposed role in the oligomerization. The formation of a zipper-like structure in X(P) is also in accordance with its existence in the aggregates of several short amyloidogenic peptides. The computed values of translational (D(T)) and rotational (D(R)) diffusion constants of 0.63 × 10(-6) cm(2)/s and 0.035 ns(-1), respectively, for this dimer are supported by the corresponding values of the Aβ42 monomer. These simulations have also elucidated several other key structural properties of these peptides. This information will be very useful to design small molecules for the inhibition and disruption of the critical Aβ42 dimers.

Methyltransferases do not work by compression, cratic, or desolvation effects, but by electrostatic preorganization

The enzyme catechol O‐methyltransferase (COMT) catalyzes the transfer of a methyl group from S‐adenosylmethionine to dopamine and related catechols. The search for the origin of COMT catalysis has led to different proposals and hypothesis, including the entropic, the NAC, and the compression proposals as well as the more reasonable electrostatic idea. Thus, it is important to understand the catalytic power of this enzyme and to examine the validity of different proposals and in particular the repeated recent implication of the compression idea. The corresponding analysis should be done by well‐defined physically‐based considerations that involve computations rather than circular interpretations of experimental results. Thus, we explore here the origin of the catalytic efficiency of COMT by using the empirical valence bond and the linear response approximation approaches. The results demonstrate that the catalytic effect of COMT is mainly due to electrostatic preorganization effects. It is also shown that the compression, NAC and entropic proposals do not account for the catalytic effect. Proteins 2015; 83:318–330.

Simulating the pulling of stalled elongated peptide from the ribosome by the translocon

The nature of the coupling between the stalling of the elongated nascent peptide chain in the ribosome and its insertion through the translocon is analyzed, focusing on the recently discovered biphasic force that overcomes the stalling barrier. The origin of this long-range coupling is explored by coarse-grained simulations that combine the translocon (TR) insertion profile and the effective chemical barrier for the extension of the nascent chain in the ribosome. Our simulation determined that the inserted H segment is unlikely to climb the TR barrier in parallel with the peptide synthesis chemical step and that the nascent chain should first overcome the chemical barriers and move into the ribosome–TR gap region before the insertion into the TR tunnel. Furthermore, the simulations indicate that the coupled TR-chemistry free energy profile accounts for the biphasic force. Apparently, although the overall elongation/insertion process can be depicted as a tug-of-war between the forces of the TR and the ribosome, it is actually a reflection of the combined free-energy landscape. Most importantly, the present study helps to relate the experimental observation of the biphasic force to crucial information about the elusive path and barriers of the TR insertion process.

Translational, rotational and internal dynamics of amyloid β-peptides (Aβ40 and Aβ42) from molecular dynamics simulations

In this study, diffusion constants [translational (D(T)) and rotational (D(R))], correlation times [rotational (tau(rot)) and internal (tau(int))], and the intramolecular order parameters (S(2)) of the Alzheimer amyloid-beta peptides Abeta40 and Abeta42 have been calculated from 150 ns molecular dynamics simulations in aqueous solution. The computed parameters have been compared with the experimentally measured values. The calculated D(T) of 1.61 x 10(-6) cm(2)/s and 1.43 x 10(-6) cm(2)/s for Abeta40 and Abeta42, respectively, at 300 K was found to follow the correct trend defined by the Debye-Stokes-Einstein relation that its value should decrease with the increase in the molecular weight. The estimated D(R) for Abeta40 and Abeta42 at 300 K are 0.085 and 0.071 ns(-1), respectively. The rotational (C(rot)(t)) and internal (C(int)(t)) correlation functions of Abeta40 and Abeta42 were observed to decay at nano- and picosecond time scales, respectively. The significantly different time decays of these functions validate the factorization of the total correlation function (C(tot)(t)) of Abeta peptides into C(rot)(t) and C(int)(t). At both short and long time scales, the Clore-Szabo model that was used as C(int)(t) provided the best behavior of C(tot)(t) for both Abeta40 and Abeta42. In addition, an effective rotational correlation time of Abeta40 is also computed at 18 degrees C and the computed value (2.30 ns) is in close agreement with the experimental value of 2.45 ns. The computed S(2) parameters for the central hydrophobic core, the loop region, and C-terminal domains of Abeta40 and Abeta42 are in accord with the previous studies.

Torque, chemistry and efficiency in molecular motors: a study of the rotary-chemical coupling in F1-ATPase.

Detailed understanding of the action of biological molecular machines must overcome the challenge of gaining a clear knowledge of the corresponding free-energy landscape. An example for this is the elucidation of the nature of converting chemical energy to torque and work in the rotary molecular motor of F1-ATPase. A major part of the challenge involves understanding the rotary–chemical coupling from a non-phenomenological structure/energy description. Here we focused on using a coarse-grained model of F1-ATPase to generate a structure-based free-energy landscape of the rotary–chemical process of the whole system. In particular, we concentrated on exploring the possible impact of the position of the catalytic dwell on the efficiency and torque generation of the molecular machine. It was found that the experimentally observed torque can be reproduced with landscapes that have different positions for the catalytic dwell on the rotary–chemical surface. Thus, although the catalysis is undeniably required for torque generation, the experimentally observed position of the catalytic dwell at 80° might not have a clear advantage for the force generation by F1-ATPase. This further implies that the rotary–chemical couplings in these biological motors are quite robust and their efficiencies do not depend explicitly on the position of the catalytic dwells. Rather, the specific positioning of the dwells with respect to the rotational angle is a characteristic arising due to the structural construct of the molecular machine and might not bear any clear connection to the thermodynamic efficiency for the system.

Theoretical insights into the functioning of metallopeptidases and their synthetic analogues.

CONSPECTUS: The selective hydrolysis of a peptide or amide bond (-(O═)C-NH-) by a synthetic metallopeptidase is required in a wide range of biological, biotechnological, and industrial applications. In nature, highly specialized enzymes known as proteases and peptidases are used to accomplish this daunting task. Currently, many peptide bond cleaving enzymes and synthetic reagents have been utilized to achieve efficient peptide hydrolysis. However, they possess some serious limitations. To overcome these inadequacies, a variety of metal complexes have been developed that mimic the activities of natural enzymes (metallopeptidases). However, in comparison to metallopeptidases, the hydrolytic reactions facilitated by their existing synthetic analogues are considerably slower and occur with lower catalytic turnover. This could be due to the following reasons: (1) they lack chemical properties of amino acid residues found within enzyme active sites; (2) they contain a higher metal coordination number compared with naturally occurring enzymes; and (3) they do not have access to second coordination shell residues that provide substantial rate enhancements in enzymes. Additionally, the critical structural and mechanistic information required for the development of the next generation of synthetic metallopeptidases cannot be readily obtained through existing experimental techniques. This is because most experimental techniques cannot follow the individual chemical steps in the catalytic cycle due to the fast rate of enzymes. They are also limited by the fact that the diamagnetic d(10) Zn(II) center is silent to electronic, electron spin resonance, and (67)Zn NMR spectroscopies. Therefore, we have employed molecular dynamics (MD), quantum mechanics (QM), and hybrid quantum mechanics/molecular mechanics (QM/MM) techniques to derive this information. In particular, the role of the metal ions, ligands, and microenvironment in the functioning of mono- and binuclear metal center containing enzymes such as insulin degrading enzyme (IDE) and bovine lens leucine aminopeptidase (BILAP), respectively, and their synthetic analogues have been investigated. Our results suggested that in the functioning of IDE, the chemical nature of the peptide bond played a role in the energetics of the reaction and the peptide bond cleavage occurred in the rate-limiting step of the mechanism. In the cocatalytic mechanism used by BILAP, one metal center polarized the scissile peptide bond through the formation of a bond between the metal and the carbonyl group of the substrate, while the second metal center delivered the hydroxyl nucleophile. The Zn(N3) [Zn(His, His, His)] core of matrix metalloproteinase was better than the Zn(N2O) [Zn(His, His, Glu)] core of IDE for peptide hydrolysis. Due to the synergistic interaction between the two metal centers, the binuclear metal center containing Pd2(μ-OH)([18]aneN6)](4+) complex was found to be ∼100 times faster than the mononuclear [Pd(H2O)4](2+) complex. A successful small-molecule synthetic analogue of a mononuclear metallopeptidase must contain a metal with a strong Lewis acidity capable of reducing the pKa of its water ligand to less than 7. Ideally, the metal center should include three ligands with low basicity. The steric effects or strain exerted by the microenvironment could be used to weaken the metal-ligand interactions and increase the activity of the metallopeptidase.

Which One Among Aspartyl Protease, Metallopeptidase, and Artificial Metallopeptidase is the Most Efficient Catalyst in Peptide Hydrolysis?

In this comparative DFT study, the hydrolysis of a peptide bond (Phe1-Phe2) by the following three types of catalysts has been studied: (1) beta-secretase (BACE2), (2) matrix metalloproteinase (MMP) and insulin degrading enzyme (IDE), and (3) [Pd(H(2)O)(4)](2+) (I(MPC)) and [Pd(2)(mu-OH)([18]aneN(6))](3+) (I(DPC)). The computed energetics predict that among these catalysts, the Zn(2+) metal center containing MMP is the most efficient in catalyzing this reaction. The two active site aspartate residues containing BACE2 catalyze this reaction with 5.0 kcal/mol higher barrier than MMP. The substitution of a His ligand with Glu in the metal center of MMP generates the active site of IDE that catalyzes the reaction with a 6.9 kcal/mol higher barrier than MMP. Both artificial peptidases I(MPC) and I(DPC) catalyze this reaction with significantly high barriers of 35.4 and 31.0 kcal/mol, respectively. The computed energetics of all the catalysts are in line with the available experimental and theoretical data.

On the Challenge of Exploring the Evolutionary Trajectory from Phosphotriesterase to Arylesterase Using Computer Simulations

The ability to design effective enzymes presents a fundamental challenge in biotechnology and also in biochemistry. Unfortunately, most of the progress on this field has been accomplished by bringing the reactants to a reasonable orientation relative to each other, rather than by rational optimization of the polar preorganization of the environment, which is the most important catalytic factor. True computer based enzyme design would require the ability to evaluate the catalytic power of designed active sites. This work considers the evolution from a phosphotriesterase (with the paraoxon substrate) to arylesterase (with the 2-naphthylhexanoate (2NH) substrate) catalysis. Both the original and the evolved enzymes involve two zinc ions and their ligands, making it hard to obtain a reliable quantum mechanical description and then to obtain an effective free energy sampling. Furthermore, the options for the reaction path are quite complicated. To progress in this direction we started with DFT calculations of the energetics of different mechanistic options of cluster models and then used the results to calibrate empirical valence bond (EVB) models and to generate properly sampled free energy surfaces for different mechanisms in the enzyme. Interestingly, it is found that the catalytic effect depends on the Zn-Zn distance making the mechanistic analysis somewhat complicated. Comparing the activation barriers of paraoxon and the 2NH ester at the beginning and end of the evolutionary path reproduced the observed evolutionary trend. However, although our findings provide an advance in exploring the nature of promiscuous enzymes, they also indicate that modeling the reaction mechanism in the case of enzymes with a binuclear zinc center is far from trivial and presents a challenge for computer-aided enzyme design.

Elucidation of Interactions of Alzheimer Amyloid β Peptides (Aβ40 and Aβ42) with Insulin Degrading Enzyme: A Molecular Dynamics Study

In this study, interactions of the two full-length Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with the fully active form of insulin degrading enzyme (IDE) through unrestrained, all-atom MD simulations have been investigated. This enzyme is a Zn-containing metallopeptidase that catalyzes the degradation of the monomeric forms of these peptides, and this process is critical for preventing the progression of Alzheimers disease (AD). The available X-ray structures of the free and small fragment-bound (Asp1-Glu3 and Lys16-Asp23 of Abeta40 and Asp1-Glu3 and Lys16-Glu22 of Abeta42) mutated forms of IDE and NMR structures of the full-length Abeta40 and Abeta42 have been used to build the starting structures for these simulations. The most representative structures derived from the Abeta40-IDE and Abeta42-IDE simulations accurately reproduced the locations of the active site Zn(2+) metal and small fragments of the substrates and their interactions with the enzyme from the X-ray structures. The remaining fragments of both the substrates were found to interact with IDE through several hydrogen bonding, pi-pi, CH-pi, and NH-pi interactions. In comparison to Abeta40, Abeta42 is more flexible and interacts through a smaller number (17-22) of hydrogen bonds in the catalytic chamber of IDE. Both the substrates adopted more beta-sheet character in the IDE environment, an observation that is in line with experiments. Their structural characteristics inside IDE are significantly different than the ones observed in aqueous solution. The atomistic level details provided by these simulations can help in the elucidation of binding and degrading mechanisms of the Abeta peptides by IDE.