Why Does Asn71 Deamidate Faster Than Asn15 in the Enzyme Triosephosphate Isomerase? Answers from Microsecond Molecular Dynamics Simulation and QM/MM Free Energy Calculations

Abstract

Deamidation is the uncatalyzed process by which asparagine or glutamine can be transformed into aspartic acid or glutamic acid, respectively. In its active homodimeric form, mammalian triosephosphate isomerase (TPI) contains two deamidation sites per monomer. Experimental evidence shows that the primary deamidation site (Asn71-Gly72) deamidates faster than the secondary deamidation site (Asn15-Gly16). To evaluate the factors controlling the rates of these two deamidation sites in TPI, we have performed graphics processing unit-enabled microsecond long molecular dynamics simulations of rabbit TPI. The kinetics of asparagine dipeptide and two deamidation sites in mammalian TPI are also investigated using quantum mechanical/molecular mechanical tools with the umbrella sampling technique. Analysis of the simulations has been performed using independent global and local descriptors that can influence the deamidation rates: desolvation effects, backbone acidity, and side chain conformations. Our findings show that all the descriptors add up to favor the primary deamidation site over the secondary one in mammalian TPI: Asn71 deamidates faster because it is more solvent accessible, the adjacent glycine NH backbone acidity is enhanced, and the Asn side chain has a preferential near attack conformation. The crucial impact of the backbone amide acidity of the adjacent glycine on the deamidation rate is shown by kinetic analysis. Our findings also shed light on the effect of high-order structure on deamidation: the deamidation in a small peptide is favored first because of the higher reactivity of the asparagine residue and then because of the stronger stability of the tetrahedral intermediate.

Biopharmaceutical Informatics: supporting biologic drug development via molecular modelling and informatics

“…However, N71 deamidates approximately twofold faster than N15 . Ugur et al . suggested that N71 deamidates faster than N15 not only due to a greater solvent exposure, but also due to higher acidity of the G72 backbone NH group, and increased ability to access reactive conformations conducive to the nucleophilic attack by the backbone nitrogen atom of G72.…”

SemiEmpirical Born-Oppenheimer Molecular Dynamics (SEBOMD) Within the Amber Biomolecular Package

“…We show in Figure 2 the average speed (in ps/day) of NVT SEBOMD simulations for different sizes of pure water molecular systems. From left to right, the systems respectively contain 64, 125, 216, or 512 water molecules, encaged in a periodic box with a density of 1.0 g/cm 3 . The D&C algorithm uses 1 water molecule per core, a first buffer of 6Å and no second buffer.…”

Molecular dynamics simulations of apo, holo, and inactivator bound GABA‐at reveal the role of active site residues in PLP dependent enzymes

“…A time step of 2.0 fs was used by the implementation of the SHAKE algorithm for the bonds involving hydrogens . The equilibration of the samples was performed in a five stage process . First, after a short 1000 step minimization, a 100 ps molecular dynamics was performed at 10 K with a strong temperature coupling (i.e., velocities were randomly updated every 10 steps in the Andersen thermostat) and with a harmonic restraint of 50 kcal/mol/Å 2 on all nonhydrogen atoms to ensure a proper geometry of the hydrogen atoms that were added by the tLEAP program and to relax the hydrogen bond network between the water molecules and the protein.…”