Enzymes and Transition State Structures Relating to SAM Metabolism
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The methionine cycle is responsible the synthesis of S-adenosyl-L-methionine (SAM), for use as a one-carbon donor for a wide variety of methyl-transfer reactions including histone and DNA methylation, as well as a number of other metabolic reactions. These metabolic pathways are essential for rapid cellular growth, meaning that small-molecule inhibitors of essential enzymes in this pathway are likely to have applications as anti-cancer therapeutics. Here we have investigated the properties of transition-state analogues of human 5"-methylthioadenosine phosphorylase (MTAP) as well as the transition-state structure of human methionine adenosyl transferase 2A (MAT2A), both of which are critical enzymes in methionine metabolism, to determine the viability of transition-state analogues of these enzymes as anti-cancer therapies.;In chapter 2 we report a new assay using 2-amino-5"-methylthioadenosine (2AMTA) as an alternative substrate for MTAP. Phosphorolysis of 2AMTA forms 2,6-diamiopurine, a fluorescent and easily quantitated product. We kinetically characterize 2AMTA with human MTAP, bacterial 5"methylthioadenosine nucleosidase MTANs and use 2,6-diaminopurine as a fluorescent substrate for yeast adenine phosphoribosyltransferase. 2AMTA was used as the substrate to kinetically characterize the dissociation constants for three transition-state analogue inhibitors of MTAP and MTAN. This assay is suitable for inhibitor screening by detection of fluorescent product, for quantitative analysis of hits by rapid and accurate measurement of inhibition constants in continuous assays, and for pre-steady-state kinetic analysis of the target enzymes.;In chapter 3 we report that MTAP transition-state analogue binding exhibits a negative heat capacity change (--DeltaCp), indicating that changes in enthalpy and entropy upon binding are strongly temperature-dependent. The DeltaCp of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Temperature dependence of pre-steady-state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition-state formation (--DeltaCp‡). A comparison of the DeltaCp‡ for MTAP pre-steady-state chemistry and DeltaCp for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition-state.;In chapter 4 we investigated oral MTDIA as an agent to treat intestinal adenomas in ApcMin/+ mice. This model uses immunocompetent mice with a high probability of intestinal adenoma formation without an inciting biological insult or additional spontaneous mutations. Mice treated with oral MTDIA had a twofold increase in life span compared to control mice. Oral MTDIA has a significant therapeutic benefit to ApcMin/+ mice.;Chapter 5 uses kinetic isotope effect (KIE), commitment factor (Cf), and binding isotope effect (BIE) measurements, combined with quantum mechanics and molecular mechanics (QM/MM) calculations, to solve the transition-state structure of MAT2A, the enzyme in humans that synthesizes S-adenosyl-Lmethionine from ATP and methionine. The enzyme forms an advanced SN2 type transition-state with a 2.035 A nucleophilic C-S bond (bond order of 0.668), and a 2.320 A leaving group C-0 bond (bond order of 0.227).;This thesis lays the groundwork for the discovery of MAT2A inhibitors, which may have anti-cancer effects when used in combination with MTAP transition state analogues.
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