Spatio-temporal correlations can drastically change the response of a MAPK pathway

Multisite covalent modification of proteins is omnipresent in eukaryotic cells. A well-known example is the mitogen-activated protein kinase (MAPK) cascade, where in each layer of the cascade a protein is phosphorylated at two sites. It has long been known that the response of a MAPK pathway strongly depends on whether the enzymes that modify the protein act processively or distributively: distributive mechanism, in which the enzyme molecules have to release the substrate molecules in between the modification of the two sites, can generate an ultrasensitive response and lead to hysteresis and bistability. We study by Green's Function Reaction Dynamics, a stochastic scheme that makes it possible to simulate biochemical networks at the particle level and in time and space, a dual phosphorylation cycle in which the enzymes act according to a distributive mechanism. We find that the response of this network can differ dramatically from that predicted by a mean-field analysis based on the chemical rate equations. In particular, rapid rebindings of the enzyme molecules to the substrate molecules after modification of the first site can markedly speed up the response, and lead to loss of ultrasensitivity and bistability. In essence, rapid enzyme-substrate rebindings can turn a distributive mechanism into a processive mechanism. We argue that slow ADP release by the enzymes can protect the system against these rapid rebindings, thus enabling ultrasensitivity and bistability

Ornithine decarboxylase activity, a clinical biomarker for evaluating cancer chemopreventive efficacy of phytomolecules

Biomarkers of cancer have made a strong traipse in predicting the disease pattern and contributed significantly to the understanding of tumour state, progression, characteristics and response to therapies. Polyamines such as putrescine, spermine and spermidine are cationic biomolecules essential for the cell cycle function and serve as excellent biomarkers of tumour progression. The polyamine biosynthesis is tightly regulated by ornithine decarboxylase, a highly inducible enzyme specific to pH, temperature, time and substrate concentration. The expression of this enzyme is very high during cell transformation and tumour progression leading to elevated level of polyamines. To measure the activity of ornithine decarboxylase an improved, easy, simple, reliable and cost-effective method has been developed utilizing small quantity of chemicals. The methodology is based on the cognizance that enzyme transforms L-ornithine hydrochloride substrate to a yellow coloured product putrescine soluble in pentanol, the absorbance of which was measured spectrophotometrically. The procedure is being utilized for evaluating cancer chemopreventive efficacy of phytomolecules. We have analyzed hundreds of molecules belonging to flavonoid, terpenes and alkaloid groups and very few were found to inhibit enzyme activity in a concentration dependent manner (0.4-50µg/mL). In addition, the molecules were also tested for their radical scavenging properties. Our results depict that molecules having phenolic groups and lactone rings in their structure are better inhibitors than their counterparts. The comparative analysis of the groups reassures flavonoids as better scavengers of radical formation and a positive correlation was observed among the nitric oxide and 2, 2-diphenyl-1-picrylhydrazyl inhibition (p<0.01). Further evaluation and augmentation may reveal novel ornthine decarboxylase inhibitors and cancer chemopreventive agents from plants

DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate.

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event

Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and Enhanced by Non-Native Small-Molecule Cofactors

Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that a non-heme iron enzyme can catalyze olefin aziridination and nitrene C–H insertion, and that these activities can be improved by directed evolution. The non-heme iron center allows for facile modification of the primary coordination sphere by addition of metal-coordinating molecules, enabling control over enzyme activity and selectivity using small molecules

Single Molecule Michaelis-Menten Equation beyond Quasi-Static Disorder

The classic Michaelis-Menten equation describes the catalytic activities for ensembles of enzyme molecules very well. But recent single-molecule experiment showed that the waiting time distribution and other properties of single enzyme molecule are not consistent with the prediction based on the viewpoint of ensemble. It has been contributed to the slow inner conformational changes of single enzyme in the catalytic processes. In this work we study the general dynamics of single enzyme in the presence of dynamic disorder. We find that at two limiting cases, the slow reaction and nondiffusion limits, Michaelis-Menten equation exactly holds although the waiting time distribution has a multiexponential decay behaviors in the nondiffusion limit.Particularly, the classic Michaelis-Menten equation still is an excellent approximation other than the two limits.Comment: 10 pages, 1 figur

Can we always sweep the details of RNA-processing under the carpet?

RNA molecules follow a succession of enzyme-mediated processing steps from transcription until maturation. The participating enzymes, for example the spliceosome for mRNAs and Drosha and Dicer for microRNAs, are also produced in the cell and their copy-numbers fluctuate over time. Enzyme copy-number changes affect the processing rate of the substrate molecules; high enzyme numbers increase the processing probability, low enzyme numbers decrease it. We study different RNA processing cascades where enzyme copy-numbers are either fixed or fluctuate. We find that for fixed enzyme-copy numbers the substrates at steady-state are Poisson-distributed, and the whole RNA cascade dynamics can be understood as a single birth-death process of the mature RNA product. In this case, solely fluctuations in the timing of RNA processing lead to variation in the number of RNA molecules. However, we show analytically and numerically that when enzyme copy-numbers fluctuate, the strength of RNA fluctuations increases linearly with the RNA transcription rate. This linear effect becomes stronger as the speed of enzyme dynamics decreases relative to the speed of RNA dynamics. Interestingly, we find that under certain conditions, the RNA cascade can reduce the strength of fluctuations in the expression level of the mature RNA product. Finally, by investigating the effects of processing polymorphisms we show that it is possible for the effects of transcriptional polymorphisms to be enhanced, reduced, or even reversed. Our results provide a framework to understand the dynamics of RNA processing

Resolution of Joint Molecules by RuvABC and RecG Following Cleavage of the Escherichia coli Chromosome by EcoKI

DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions.</p

Stochastic theory of large-scale enzyme-reaction networks: Finite copy number corrections to rate equation models

Chemical reactions inside cells occur in compartment volumes in the range of atto- to femtolitres. Physiological concentrations realized in such small volumes imply low copy numbers of interacting molecules with the consequence of considerable fluctuations in the concentrations. In contrast, rate equation models are based on the implicit assumption of infinitely large numbers of interacting molecules, or equivalently, that reactions occur in infinite volumes at constant macroscopic concentrations. In this article we compute the finite-volume corrections (or equivalently the finite copy number corrections) to the solutions of the rate equations for chemical reaction networks composed of arbitrarily large numbers of enzyme-catalyzed reactions which are confined inside a small sub-cellular compartment. This is achieved by applying a mesoscopic version of the quasi-steady state assumption to the exact Fokker-Planck equation associated with the Poisson Representation of the chemical master equation. The procedure yields impressively simple and compact expressions for the finite-volume corrections. We prove that the predictions of the rate equations will always underestimate the actual steady-state substrate concentrations for an enzyme-reaction network confined in a small volume. In particular we show that the finite-volume corrections increase with decreasing sub-cellular volume, decreasing Michaelis-Menten constants and increasing enzyme saturation. The magnitude of the corrections depends sensitively on the topology of the network. The predictions of the theory are shown to be in excellent agreement with stochastic simulations for two types of networks typically associated with protein methylation and metabolism.Comment: 13 pages, 4 figures; published in The Journal of Chemical Physic