Recent Advances in Life Sciences

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Kinetics of Sequential Enzyme Reactions and Electrostatic Channeling
Peter M. Kekenes-Huskey, Vincent T. Metzger, J. Andrew McCammon ( University of California at San Diego, San Diego, CA, USA)

In cells, many enzyme-catalyzed reactions are coupled as a series of biochemical steps. Thus, the reaction rate is dependent on the kinetics of intermediate reaction steps. In this work, we study the case of two coupled enzyme reactions, where the product of the first reaction serves as the substrate of the second reaction. Using a diffusion-limited reaction model, we demonstrate that the overall kinetics is strongly dependent on the separation distance between two enzymes, although interestingly, we find that in some cases, the maximal rate occurs at a finite, non-smallest separation between enzymes. This is because the second enzyme in proximity can block the access of substrate for the first enzyme reaction, which leads to the decrease of product of the first reaction (substrate for the second reaction). We further demonstrate how this reaction rate is additionally dependent on the nature of electrostatic interactions between reactants and the two enzymes. We demonstrate the interplay of these concepts for the dihydrofolate reductase-thymidylate synthase (DHFR-TS) systems, whereby methylene tetrahydrofolate is converted to tetrahydrofoloate. This study suggests the role of species-specific electrostatic and geometric factors in optimizing reaction rates of substrate-channeling systems.

Single Molecule Force Spectroscopy Reveals Force-Enhanced Binding of Calcium Ions by Gelsolin
Chunmei Lv, Xiang Gao, Wenfei Li (Nanjing University, Nanjing, China)

Force is increasingly recognized as an important element in controlling biological processes. Forces are able to deform native protein conformations leading to protein-specific effects. Although in a few cases, the exposure of novel protein binding sites by force has been reported, mechanical stress generally leads to the decrease of protein-protein binding affinities. In this paper, we demonstrate that the calcium-binding affinity of the actin-binding protein gelsolin domain G6 is enhanced, rather than decreased, by mechanical force. Using a recently developed single molecule-binding assay based on atomic force microscopy, it establish that the calcium-binding affinity of G6 increases exponentially with the applied force, up to the point of G6 unfolding. This implies that gelsolin will be activated at lower calcium ion levels when subjected to tensile forces and suggests a basis for enhanced cooperativity during multi-cation induced activation. This is, to our knowledge, the first experimental verification of the force-amplified ligand binding concept at the molecular level. Such force-amplified ligand binding is fundamentally different from the widely accepted ideas of “catch bond” and force activated binding. The demonstration that cation-protein binding affinities can be force-dependent provides a new paradigm in understanding the complex interactions of cation-regulated proteins in stressful cellular environments, such as those found in the cytoskeleton-rich leading edge and at cell adhesions.

Insights into the Inhibition Mechanism of Biomolecular Self-Assembly from Chemical Kinetics:
Michele Vendruscolo, Christopher M. Dobson, Tuomas P.J. Knowles: (University of Cambridge, Cambridge, United Kingdom)

Understanding and control the aggregation of biomolecules at the molecular level can open attractive possibilities to correct dysfunctional cell behaviour. For instance, the inhibition of protein aggregation is emerging as a potential attractive therapeutic strategy against several neurodegenerative disorders. For the development of successful treatments, it is crucial to achieve a controlled intervention on specific toxic species. In this perspective, an understanding of the molecular inhibition mechanism of protein self-assembly is of fundamental importance but remains challenging to achieve. In this work, we demonstrate how chemical kinetic analysis can be applied to elucidate the molecular mechanism of inhibition of several classes of compounds such as small chemical molecules, nanoparticles, peptides and proteins. By applying a population balance model we show how it is possible to obtain information on the specific inhibited microscopic event and on the specific protein target species responsible for this inhibition. We demonstrate the potentiality of the approach by analyzing the inhibition mechanism of selected chaperones, protein regulators of the proteostasis network and relevant naturally occurring inhibitors of protein aggregation, on the aggregation of a yeast prion protein and of Abeta42, the peptide involved in Alzheimer’s disease. In addition, we discuss relevant implications of the controlled inhibition of protein aggregation in the engineering of the fibrillation reaction pathway and in the development of effective therapeutic strategies.

Filament Assembly by Phosphofructokinase-1, the Gatekeeper of Glycolysis:
Bradley Webb, Larry Ackerman, Diane Barber. Cell and Tissue Biology, UCSF, San Francisco, CA, USA

The cytoskeleton is conventionally viewed as being composed of three filamentous networks; microfilaments, microtubules, and intermediate filaments. This view is challenged by the findings that metabolic enzymes can form filaments with structural functions. We report that phosphofructokinase-1 (PFK1), the first rate-limiting step of glycolysis, assembles into filaments in vitro and in cells. Transmission electron microscopy (TEM) showed that purified liver PFK1 is mainly tetrameric and occasionally formed short filaments in the absence of substrate. Adding the substrate fructose 6-phosphate (F6P) induced the assembly of predominantly long filaments measuring up to 250nm. PFK1 filaments were less rigid than actin polymers, displaying right angles in contiguous assemblies. The filaments were composed of individual tetramers and had a uniform 11 nm width, resembling an organized addition of subunits forming polymers. Regulated assembly into filaments was also indicated by light scattering measurements that showed a rapid substrate dependent increase in scattering followed by a stable plateau. Increased light scattering was blocked by excess ATP, which inhibits PFK1 activity. To further confirm activity-dependent filament assembly we generated an inactive but tetrameric liver PFK1 mutant, His199Tyr, and found that in the presence of F6P it does not form filaments, as determined by TEM, or show an increase in light scattering. To assess filament formation by PFK1 in cells, expressed GFP-tagged PFK1 and used live-cell imaging to examine GFPPFK1 dynamics. Confocal microscopy revealed that cytosolic PFK1 was recruited to the distal margin of lamellipodia that were devoid of mitochondria. TIRF microscopy revealed that GFP-PFK1 formed dynamic punctate. These data indicate that active but not inactive PFK1 assembles into tetramer-aligned filaments. The activity-dependent recruitment and assembly of PFK1 filaments at the plasma membrane could provide a scaffolding and structural framework for localized ATP production in lamellipodia that lack mitochondria.

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