Stanislaw T. Wlodek

Acetylcholinesterase: Role of the enzyme's charge distribution in steering charged ligands toward the active site

The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42 A radius centered in the enzyme monomer and separated from the protein surface by 1-14 A is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32 A radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a neutral ligand. However, the differences among the encounter rates for the various mutants considered here are not pronounced, all rate constants being within +/- 10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzymes total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzymes charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it has entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase.

Binding of Cations and Protons in the Active Site of Acetylcholinesterase

The active site of acetylcholinesterase contains a number of ionizable residues. The pK as of the catalytic histidine, His 440, is believed to be 6.3, based upon enzyme kinetic studies. However, the pK as of the other residues have not been measured. Here, we describe calculations of the pK a of the ionizable groups in this enzyme. Interestingly, the initial calculations predict a pK a of 9.3 for His 440. The deviation of 3 pK a units from the measured pK a is traceable to the influence of Glu 199 and Glu 443 upon His 440. We argue that the deviation does not represent a failure of the computational method. Rather it points to the need for an adjustment in the model of the protein. The adjustment we suggest involves a monovalent cation bound in the active site, near Glu 199 and His 440. Including such a cation in the calculations brings the computed pK a of His 440 into agreement with the measured value. Furthermore, the idea that a bound cation substantially reduces tbe pK a of His 440 leads to satisfying explanations of a number of otherwise puzzling experimental data.

Prediction of pKas of Titratable Residues in Proteins Using a Poisson-Boltzmann Model of the Solute-Solvent System

This article provides an overview of an algorithm used for the prediction of ionization constants of titratable residues in proteins. The algorithm is based on an assumption that the difference in protonation behavior of a given group in an isolated state in solution, for which the ionization constant is assumed to be known, and the protonation behavior in the protein environment is purely electrostatic in origin. Calculations of the relevant electrostatic free energies are based on the Poisson-Boltzmann (PB) model of the protein-solvent system and the finitedifference solution to the corresponding PB equation. The resultant multiple site titration problem is treated by one of two methods. The first is a hybrid approach, based on collecting ionizable groups into clusters. The second method is a Monte Carlo approach based on the Metropolis algorithm for extracting a sufficient number of low-energy ionization states out of all possible states, to obtain a correct estimation of thermodynamic properties of the system.

Computer Modeling of Acetylcholinesterase and Acetylcholinesterase-Ligand Complexes

Site directed mutagenesis and computer simulations studies have become valuable tools for the understanding of function-structure relations of proteins. At the current level of computer technology, the quantitative agreement between experimental and molecular simulations results is not always satisfactory for proteins of acetylcholinesterase (AChE) size and complexity. However, the comparison of both types of results can increase dramatically our knowledge on molecular mechanisms of protein action.

Molecular Dynamics of Acetylcholinesterase Dimer

The dynamic properties of acetylcholinesterase dimer from Torpedo californica liganded with tacrine (TcAChE-THA) have been studied using molecular dynamics (MD). 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, suggesting that transport of solvent molecules participating in catalysis can occur across the porous wall, contributing to the efficiency of the enzyme. A large scale motion of slight contraction and relative rotation of the protein subunits has been detected.

COMPUTER SIMULATION STUDIES OF ACETYLCHOLINESTERASE DYNAMICS AND ACTIVITY

Computer simulations of the activity of acetylcholinesterase (AChE) are shedding light on the origins of the selectivity, mechanism, and efficiency of the enzyme. In the following, a brief discussion is presented on two aspects of the the enzymatic activity: the role of the electrostatic field of the enzyme in speeding its binding of cationic substrates and inhibitors, and the role of fluctuations in the structure of the enzyme in facilitating this binding.

I/O Limitations in Parallel Molecular Dynamics

We discuss data production rates and their impact on the performance of scientific applications using parallel computers. On one hand, too high rates of data production can be overwhelming, exceeding logistical capacities for transfer, storage and analysis. On the other hand, the rate limiting step in a computationally-based study should be the human-guided analysis, not the calculation. We present performance data for a biomolecular simulation of the enzyme, acetylcholinesterase, which uses the parallel molecular dynamics program EulerGROMOS. The actual production rates are compared against a typical time frame for results analysis where we show that the rate limiting step is the simulation, and that to overcome this will require improved output rates.