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Aspartates

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George D Markham – One of the best experts on this subject based on the ideXlab platform.

  • the bifunctional active site of s adenosylmethionine synthetase roles of the active site Aspartates
    Journal of Biological Chemistry, 1999
    Co-Authors: John C Taylor, George D Markham

    Abstract:

    Abstract S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPPi) from ATP, and subsequently PPPi is hydrolyzed to PPi and Pibefore product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg2+ ions (* denotes a residue from a second subunit); Aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k cat reductions of ∼103-fold in AdoMet synthesis, whereas the MgATP and methionine K m values change by less than 3- and 8-fold, respectively. In the partial reaction of PPPihydrolysis, mutants of the Mg2+ binding residues have >700-fold reduced catalytic efficiency (k cat/K m), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPPi hydrolysis by Mg2+ site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPPi hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca2+ uniquely activates the D271A mutant enzyme to 15% of the level of Mg2+, in contrast to the ∼1% Ca2+ activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca2+ and the smaller Mg2+.

Adrian E Roitberg – One of the best experts on this subject based on the ideXlab platform.

  • ph remd simulations indicate that the catalytic Aspartates of hiv 1 protease exist primarily in a monoprotonated state
    Journal of Physical Chemistry B, 2014
    Co-Authors: Dwight T Mcgee, Jesse Edwards, Adrian E Roitberg

    Abstract:

    The protonation state of the catalytic Aspartates of HIV-1 protease (HIVPR) is atypical and as a result is the subject of much debate. Modeling of the correct protonation state of the Aspartates is vital in computational drug design. Using pH replica-exchange molecular dynamics, we simulated the apo and bound forms of HIV-1 protease with 12 different protease inhibitors to investigate the pKa of not only the catalytic dyad but also the other titrating residues in HIVPR. The results obtained from these simulations are compared to experiment where possible. This study provides evidence that the catalytic Aspartates are primarily in a monoprotonated state for both the apo and bound forms of HIVPR in the pH range where generally most experiments and computational simulations occur.

John C Taylor – One of the best experts on this subject based on the ideXlab platform.

  • the bifunctional active site of s adenosylmethionine synthetase roles of the active site Aspartates
    Journal of Biological Chemistry, 1999
    Co-Authors: John C Taylor, George D Markham

    Abstract:

    Abstract S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPPi) from ATP, and subsequently PPPi is hydrolyzed to PPi and Pibefore product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg2+ ions (* denotes a residue from a second subunit); Aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k cat reductions of ∼103-fold in AdoMet synthesis, whereas the MgATP and methionine K m values change by less than 3- and 8-fold, respectively. In the partial reaction of PPPihydrolysis, mutants of the Mg2+ binding residues have >700-fold reduced catalytic efficiency (k cat/K m), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPPi hydrolysis by Mg2+ site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPPi hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca2+ uniquely activates the D271A mutant enzyme to 15% of the level of Mg2+, in contrast to the ∼1% Ca2+ activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca2+ and the smaller Mg2+.