Enzymatic Transition State Theory and Transition State Analogue Design

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Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me- H 2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-L-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an S N 2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.enzyme mechanism | transition state structure | histone methylation | kinetic isotope effects

mentioning

confidence: 99%

mentioning

confidence: 99%

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36

Poulin

Schneck

Matico

et al. 2016

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“…ground-state analogues | enzyme mechanism T he stabilization of transition states (TS) is central to the high reaction rates achieved by enzymes (1)(2)(3). Most studies to date have focused on TSs that include a chemical transformation step, where the reacting species have some partial chemical bonding character.…”

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confidence: 99%

Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate

Griffin

Bowler

Baxter

et al. 2012

Experimental observations of fluoromagnesate and fluoroaluminate complexes of β-phosphoglucomutase (β-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of β-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.ground-state analogues | enzyme mechanism T he stabilization of transition states (TS) is central to the high reaction rates achieved by enzymes (1-3). Most studies to date have focused on TSs that include a chemical transformation step, where the reacting species have some partial chemical bonding character. However, enzymes also have to avoid substantial barriers associated with nonchemical transformation steps and hence may be required to stabilize other regions of the complex energy surface corresponding to the reaction coordinate. The concept of near attack conformers (NACs) was introduced as a useful tool to help analyze these different roles, by partitioning chemical transformation and nonchemical transformation steps (4). NACs were defined as species where the reactants have an appropriate geometry for the development of the chemical TS (5, 6). Furthermore, the component of the TS barrier ascribed to the chemical TS was proposed to be relatively constant within systems carrying out similar chemistry, on the basis of a linear relationship between the (calculated) population of NACs and the corresponding reaction rates (5). Hence, in free-energy terms, the NAC model apportions the TS barrier (ΔG † ) between a variable conformational component (ΔG NAC ) and an invariant chemical (covalent) component (ΔG chem ; Fig. 1). For some uncatalyzed reactions, it was proposed that a major component of the TS free-energy barrier is the population of NACs, t...

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confidence: 99%

Intramolecular¹³C pattern in hexoses from autotrophic and heterotrophic C₃plant tissues

Gilbert

Robins

Remaud

et al. 2012

The stable carbon isotope 13 C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against 13 CO 2 so that source sugars (photosynthates) are on average 13 C depleted by 20‰ compared with atmospheric CO 2 . The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively C abundance in their specific precursor C-atom positions. However, the intramolecular isotope pattern in source leaf glucose and the isotope fractionation associated with key enzymes involved in sugar interconversions are currently unknown. To gain insight into these, we have analyzed the intramolecular isotope composition in source leaf transient starch, grain storage starch, and root storage sucrose and measured the site-specific isotope fractionation associated with the invertase (EC 3.2.1.26) and glucose isomerase (EC 5.3.1.5) reactions. When these data are integrated into a simple steady-state model of plant isotopic fluxes, the enzyme-dependent fractionations satisfactorily predict the observed intramolecular patterns. These results demonstrate that glucose and sucrose metabolism is the primary determinant of the 13 C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals.