Insight into the phosphodiesterase mechanism from combined QM/MM free energy simulations.
ABSTRACT Molecular dynamics simulations employing a combined quantum mechanical and molecular mechanical potential have been carried out to elucidate the reaction mechanism of the hydrolysis of a cyclic nucleotide cAMP substrate by phosphodiesterase 4B (PDE4B). PDE4B is a member of the PDE superfamily of enzymes that play crucial roles in cellular signal transduction. We have determined a two-dimensional potential of mean force (PMF) for the coupled phosphoryl bond cleavage and proton transfer through a general acid catalysis mechanism in PDE4B. The results indicate that the ring-opening process takes place through an S(N)2 reaction mechanism, followed by a proton transfer to stabilize the leaving group. The computed free energy of activation for the PDE4B-catalyzed cAMP hydrolysis is about 13 kcal·mol(-1) and an overall reaction free energy is about -17 kcal·mol(-1), both in accord with experimental results. In comparison with the uncatalyzed reaction in water, the enzyme PDE4B provides a strong stabilization of the transition state, lowering the free energy barrier by 14 kcal·mol(-1). We found that the proton transfer from the general acid residue His234 to the O3' oxyanion of the ribosyl leaving group lags behind the nucleophilic attack, resulting in a shallow minimum on the free energy surface. A key contributing factor to transition state stabilization is the elongation of the distance between the divalent metal ions Zn(2+) and Mg(2+) in the active site as the reaction proceeds from the Michaelis complex to the transition state.
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ABSTRACT: Since the discovery in 1957 that cyclic AMP acts as a second messenger for the hormone adrenaline, interest in this molecule and its companion, cyclic GMP, has grown. Over a period of nearly 50 years, research into second messengers has provided a framework for understanding transmembrane signal transduction, receptor-effector coupling, protein-kinase cascades and downregulation of drug responsiveness. The breadth and impact of this work is reflected by five different Nobel prizes.Nature Reviews Molecular Cell Biology 10/2002; 3(9):710-8. · 39.12 Impact Factor
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ABSTRACT: We have investigated a means of producing thin, oriented lipid monolayers which are stable under repeated washing and which may be useful in biosensing or surface-coating applications. Phosphatidylcholine and the glycosphingolipid GM1 were used as representative lipids for this work. Initially, a mixed self-assembled monolayer of octanethiol and hexadecanethiol was produced on a gold surface. This hydrophobic monolayer was then brought into contact with a thin lipid film that had been assembled at the liquid/air interface of a solution, allowing the lipid to deposit on the gold surface through hydrophobic interactions. The lipid layer was then heated to cause intermingling of the fatty acid and alkanethiol chains and cooled to form a highly stable film which withstood repeated rinsing and solution exposure. Presence and stability of the film were confirmed via ellipsometry, Fourier transform infrared spectroscopy, and quartz crystal microbalance (QCM), with an average overall film thickness of approximately 3.5 nm. This method was then utilized to produce GM1 layers on gold-coated QCM crystals for affinity sensing trials with cholera toxin. For these sensing elements, the lower detection limit of cholera toxin was found to be approximately 0.5 microg/mL, with a logarithmic relationship between toxin concentration and frequency response spanning over several orders of magnitude. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity.Langmuir 08/2004; 20(15):6501-6. · 4.19 Impact Factor
Article: How do SET-domain protein lysine methyltransferases achieve the methylation state specificity? Revisited by Ab initio QM/MM molecular dynamics simulations.[show abstract] [hide abstract]
ABSTRACT: A distinct protein lysine methyltransferase (PKMT) only transfers a certain number of methyl group(s) to its target lysine residue in spite of the fact that a lysine residue can be either mono-, di-, or tri-methylated. In order to elucidate how such a remarkable product specificity is achieved, we have carried out ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations on two SET-domain PKMTs: SET7/9 and Rubisco large subunit methyltransferase (LSMT). The results indicate that the methylation state specificity is mainly controlled by the methyl-transfer reaction step, and confirm that SET7/9 is a mono-methyltransferase while LSMT has both mono-and di-methylation activities. It is found that the binding of the methylated lysine substrate in the active site of SET7/ 9 opens up the cofactor AdoMet binding channel so that solvent water molecules get access to the active site. This disrupts the catalytic machinery of SET7/9 for the di-methylation reaction, which leads to a higher activation barrier, whereas for the LSMT, its active site is more spacious than that of SET7/9, so that the methylated lysine substrate can be accommodated without interfering with its catalytic power. These detailed insights take account of protein dynamics and are consistent with available experimental results as well as recent theoretical findings regarding the catalytic power of SET7/9.Journal of the American Chemical Society 04/2008; 130(12):3806-13. · 9.91 Impact Factor