Gating of the Active Site of Triose Phosphate Isomerase: Brownian Dynamics Simulations of Flexible Peptide Loops in the EnzymeRebecca C. Wade, Malcolm E. Davis, Brock A. Luty, Jeffry D. Madura and J. Andrew McCammonBiophysical Journal, Vol. 64, No. 1, pp. 9-15 (1993) [PubMed 8431552]The enzyme triose phosphate isomerase has flexible peptide loops at its
active sites. The loops close over these sites upon substrate binding,
suggesting that the dynamics of the loops could be of mechanistic and
kinetic importance. To investigate these issues, the loop motions in the
dimeric enzyme were simulated by Brownian dynamics. The two loops, one
on each monomer, were represented by linear chains of appropriately
parameterized spheres, each sphere corresponding to an amino acid
residue. The loops moved in the electrostatic field of the rest of the
enzyme, which was held rigid in its crystallographically observed
conformation. In the absence of substrate, the loops exhibited gating of
the active site with a period of about 1 ns and occupied "closed"
conformations for about half of the time. As the period of gating is
much shorter than the enzyme-substrate relaxation time, the motion of
the loops does not reduce the rate constant for the approach of
substrate from its simple diffusion-controlled value. This suggests that
the flexible loops may have evolved to create the appropriate
environment for catalysis while, at the same time, minimizing the
kinetic penalty for gating the active site.
Inversion of Receptor Binding Preferences by Mutagenesis: Free Energy Thermodynamic Integration Studies on Sugar-Binding to L-Arabinose Binding ProteinsM. Zacharias, T.P. Straatsma, J.A. McCammon and F.A. QuiochoBiochemistry, Vol. 32, No. 29, pp. 7428-7434 (1993) [PubMed 8338840]The
Escherichia coli L-arabinose-binding protein (ABP)
participates as a specific receptor in the transport of L-arabinose,
D-fucose, and D-galactose through the periplasmic space. The wild-type
protein binds L-arabinose about 40 times more strongly than D-fucose. A
mutation of the protein at position 108 (Met → Leu) causes a
specificity change. The Met
108Leu ABP slightly prefers
binding of D-fucose over L-arabinose. Molecular dynamics (MD) and
thermodynamic integration (TI) computer simulations were performed to
study the mechanism of sugar discrimination and specificity change based
on the known high-resolution X-ray structures. The specificity change
was evaluated by calculating the difference in free energy of
L-arabinose versus D-fucose bound to wild-type and Met
108Leu
ABP. The calculated free energy differences are consistent with the
experimentally observed specificity of wild-type and
Met
108Leu ABP. The simulations indicate that the specificity
change of Met
108Leu is accomplished mainly by reduced
Lennard-Jones interactions of residue 108 with L-arabinose and improved
interactions with D-fucose. In addition to MD/TI calculations on sugar
binding, finite difference Poisson-Boltzmann calculations were performed
to identify the most stable ionization state of buried ionizable
residues in ABP.