Examinations of the Chemical Step in Enzyme Catalysis

Singh, Priyanka; Islam, Zahidul; Kohen, Amnon

doi:10.1016/bs.mie.2016.05.017

Cited by 16 publications

(22 citation statements)

References 88 publications

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“…While the interpretation of that isotope effect is complex, 51 it is more similar to the WT in N177S than N177D, suggesting that mutation to S, while geometrically more dramatic, has a lesser effect on the TS of the hydride transfer step. 52 More insight can be gleaned from analysis of the slope of the KIE int , as reflected by ΔEa,T−H=Ea,T−Ea,H. As is typical for WT enzymes with their usual substrates, Δ AE a , T-H for the WT TSase is essentially zero.…”

Section: Resultsmentioning

confidence: 99%

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase

et al. 2018

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Thymidylate synthase (TSase), an enzyme responsible for the de novo biosynthesis of 2’-deoxythymidine 5’-monophosphate (thymidylate, dTMP) necessary for DNA synthesis, has been a drug target for decades. TSase is a highly conserved enzyme across species ranging from very primitive organisms to mammals. Among the many conserved active site residues, an asparagine (N177, using Escherichia coli residues numbering) appears to make direct hydrogen bonds with both the C4=O4 carbonyl of the 2’-deoxyuridine 5’-monophosphate (uridylate, dUMP) substrate and its pyrimidine ring’s N3. Recent studies have reassessed the TSase catalytic mechanism, focusing on the degree of negative charge accumulation at the O4 carbonyl of the substrate during two critical H-transfers - a proton abstraction and a hydride transfer. To obtain insights into the role of this conserved N177 on the hydride transfer, we examined its aspartic acid (D) and serine (S) mutants - each of which is expected to alter hydrogen bonding and charge stabilization around the C4=O4 carbonyl of the 2’-deoxyuridine 5’-monophosphate (uridylate, dUMP) substrate. Steady-state kinetics, substrate binding order studies and temperature-dependency analysis of intrinsic KIEs for the hydride transfer step of the TSase catalytic cycle suggest the active site of N177D is not precisely organized for that step. A smaller disruption was observed for N177S, which could be rationalized by partial compensation by water molecules and rearrangement of other residues toward preparation of the system for the hydride transfer under study. These experimental findings are qualitatively mirrored by QM/MM computational simulations, thereby shedding light on the sequence and synchronicity of steps in the TSase-catalyzed reaction. This information could potentially inform the design of mechanism-based drugs targeting this enzyme.

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Section: Resultsmentioning

confidence: 99%

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase

et al. 2018

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“…A significant number of studies in the temperature dependence of rates of enzymatic catalysis reactions has inspired several formulations for the description of the mechanism involved in these processes [64][65][66][67]. Results in the kinetics of catalytic reactions of the dehydrogenase and oxidase enzymes have shown an undoubted super-Arrhenius behaviour [25,53,54,68].…”

Section: (A) Super-arrheniusmentioning

confidence: 99%

Kinetics of low-temperature transitions and a reaction rate theory from non-equilibrium distributions

Aquilanti

Coutinho

Carvalho-Silva

2017

Phil. Trans. R. Soc. A.

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relaxing the thermodynamic equilibrium limit, either for a binomial distribution if d > 0 or for a negative binomial distribution if d < 0, formally corresponding to Fermionlike or Boson-like statistics, respectively. The current status of the phenomenology is illustrated emphasizing case studies; specifically (i) the super-Arrhenius kinetics, where transport phenomena accelerate processes as the temperature increases; (ii) the sub-Arrhenius kinetics, where quantum mechanical tunnelling propitiates low-temperature reactivity; (iii) the anti-Arrhenius kinetics, where processes with no energetic obstacles are rate-limited by molecular reorientation requirements. Particular attention is given for case (i) to the treatment of diffusion and viscosity, for case (ii) to formulation of a transition rate theory for chemical kinetics including quantum mechanical tunnelling, and for case (iii) to the stereodirectional specificity of the dynamics of reactions strongly hindered by the increase of temperature.This article is part of the themed issue 'Theoretical and computational studies of nonequilibrium and non-statistical dynamics in the gas phase, in the condensed phase and at interfaces'.

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“…We observe that in all of the GS trajectories, the DAD distribution is centered around ∼4.0 ± 0.1 Å. These DADs are longer when compared to its evolved counterpart ( ec DHFR DAD = ∼3.6 ± 0.1 Å), 75 , 77 , 136 − 139 suggesting that the more evolved enzyme is better preorganized. Therefore, R67 DHFR requires significant reorganization to bring the reactants together to a reactive state.…”

Section: Resultsmentioning

confidence: 82%

Temperature-Dependent Kinetic Isotope Effects in R67 Dihydrofolate Reductase from Path-Integral Simulations

Mhashal

Major

2021

J. Phys. Chem. B

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Calculation of temperature-dependent kinetic isotope effects (KIE) in enzymes presents a significant theoretical challenge. Additionally, it is not trivial to identify enzymes with available experimental accurate intrinsic KIEs in a range of temperatures. In the current work, we present a theoretical study of KIEs in the primitive R67 dihydrofolate reductase (DHFR) enzyme and compare with experimental work. The advantage of R67 DHFR is its significantly lower kinetic complexity compared to more evolved DHFR isoforms. We employ mass-perturbation-based path-integral simulations in conjunction with umbrella sampling and a hybrid quantum mechanics–molecular mechanics Hamiltonian. We obtain temperature-dependent KIEs in good agreement with experiments and ascribe the temperature-dependent KIEs primarily to zero-point energy effects. The active site in the primitive enzyme is found to be poorly preorganized, which allows excessive water access to the active site and results in loosely bound reacting ligands.

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Examinations of the Chemical Step in Enzyme Catalysis

Cited by 16 publications

References 88 publications

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase

Kinetics of low-temperature transitions and a reaction rate theory from non-equilibrium distributions

Temperature-Dependent Kinetic Isotope Effects in R67 Dihydrofolate Reductase from Path-Integral Simulations

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