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Entropic intermediates and hidden rate-limiting steps in seemingly concerted cycloadditions. Observation, prediction, and origin of an isotope effect on recrossing.

ABSTRACT: An unusual intramolecular kinetic isotope effect (KIE) in the reaction of dichloroketene with cis-2-butene does not fit with a simple asynchronous cycloaddition transition state, but it can be predicted from trajectory studies on a bifurcating energy surface. The origin of the KIE is related to a high propensity for transition state recrossing in this system, with heavier masses recrossing less. The KIE can also be predicted by a statistical model that treats the cycloaddition as a stepwise mechanism, the rate-limiting second step being associated with an entropic barrier for formation of the second carbon-carbon bond. The relevance of this stepwise mechanism to other asynchronous but seemingly concerted cycloadditions is suggested by examination of organocatalytic Diels-Alder reactions.

SUBMITTER: Gonzalez-James OM

PROVIDER: S-EPMC3271175 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Entropic intermediates and hidden rate-limiting steps in seemingly concerted cycloadditions. Observation, prediction, and origin of an isotope effect on recrossing.

Gonzalez-James Ollie M OM Kwan Eugene E EE Singleton Daniel A DA

Journal of the American Chemical Society 20120118 4

An unusual intramolecular kinetic isotope effect (KIE) in the reaction of dichloroketene with cis-2-butene does not fit with a simple asynchronous cycloaddition transition state, but it can be predicted from trajectory studies on a bifurcating energy surface. The origin of the KIE is related to a high propensity for transition state recrossing in this system, with heavier masses recrossing less. The KIE can also be predicted by a statistical model that treats the cycloaddition as a stepwise mech ...[more]

PMID: 22229840

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Project description:1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0-30mum) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (;isotope-dilution plots'), values were obtained for (a) the V(max.) of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the V(max.) and pool concentration at a critical thymidine concentration (approx. 5mum). The bimodality was removed by amethopterin. The V(max.) determined with deoxy[U-(14)C]cytidine was approximately equal to the sum of the V(max.) determined with deoxy[5-(3)H]cytidine and the V(max.) determined with [Me-(3)H]thymidine at thymidine concentrations above 5mum. 3. The thymidine competitor pool at thymidine concentrations above 5mum was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5mum. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16mum) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5mum, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5mum, thymidine kinase limits the rate of labelling with [Me-(3)H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4mum). At thymidine concentrations greater than 5mum, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.

Project description:The folding kinetics of Rd-apocytochrome b562 is two-state, but native-state hydrogen exchange experiments show that there are discrete partially unfolded (PUF) structures in equilibrium with the native state. These PUF structures are called hidden intermediates because they are not detected in kinetic experiments and they exist after the rate-limiting step. Structures of the mimics of hidden intermediates of Rd-apocytochrome b562 are resolved by NMR. Based upon their relative stability and structural features, the folding mechanism was proposed to follow a specific pathway (unfolded ? rate-limiting transition state ? PUF1 ? PUF2 ? native). Investigating the roles of equilibrium PUF structures in folding kinetics and their interrelationship not only deepens our understanding of the details of folding mechanism but also provides guides in protein design and prevention of misfolding. We performed molecular dynamics simulations starting from a hidden intermediate and the native state of Rd-apocytochrome b562 in explicit solvent, for a total of 37.18 ?s mainly with Anton. We validated our simulations by detailed comparison with experimental data and other computations. We have verified that we sampled the post rate-limiting transition state region only. Markov state model was used to analyze the simulation results. We replace the specific pathway model with a network model. Transition-path theory was employed to calculate the net effective flux from the most unfolded state towards the most folded state in the network. The proposed sequential folding pathway via PUF1 then more stable, more native-like PUF2 is one of the routes in our network, but it is not dominant. The dominant path visits PUF2 without going through PUF1. There is also a route from PUF1 directly to the most folded state in the network without visiting PUF2. Our results indicate that the PUF states are not necessarily sequential in the folding. The major routes predicted in our network are testable by future experiments such as single molecule experiment.

Dataset Information

Entropic intermediates and hidden rate-limiting steps in seemingly concerted cycloadditions. Observation, prediction, and origin of an isotope effect on recrossing.

Publications

Entropic intermediates and hidden rate-limiting steps in seemingly concerted cycloadditions. Observation, prediction, and origin of an isotope effect on recrossing.

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