The intracellular milieu is like a bustling metropolitan city. A dizzying array of cellular components chaotically zip around in the city. Intriguingly, amidst this confusion, metabolic processes still occur efficiently. These processes are usually driven by enzymatic reactions. Enzymes process substrates and pass the resultant products for further downstream reactions. This process, known as substrate channeling, promotes specific enzymatic pathways resulting in high product yields.
How do these enzymes find each other
to carry out this cascade?
The research led by Dr. Sen used three different approaches- theoretical modelling, microfluidic channels, and fluorescence spectroscopy to address the question: “How do these enzymes find each other to carry out this cascade”? They found that chemotaxis drives the formation of an enzyme assembly line. So, what is chemotaxis? It is a phenomenon where movement is driven by the concentration gradient of specific molecules. In general, only living cells are known to carry out chemotaxis in response to a chemical stimulus.
Gradient was set up using a three-inlet/one-outlet
microfluidic device.
In the current study, the first four enzymes in glycolysis: hexokinase (HK), phosphoglucose isomerase (Iso), phosphofructokinase (PFK), and aldolase (Ald) were used to demonstrate chemotaxis of enzymes in response to a substrate gradient. The gradient was set up using a three-inlet/one-outlet microfluidic device. The three inlets of the device are the central channel, the left flanking channel, and the right flanking channel. The composition of all three inlets can be varied. Since they all feed into the same outlet channel, the migration of molecules from the central channel (for example) towards either the left or the right channel can be measured. This device therefore sets up a concentration gradient because molecules that are fed into either of the flanking channels slowly diffuse towards the central channel and vice versa. The enzymes were fluorescently labelled in order to track their movement.
Enzyme movement was first demonstrated using hexokinase (HK). D-glucose, HK, and Mg2+ were introduced into all inlets. Addition of ATP in one of the inlets completed the requirements for catalysis and caused the diffusion of HK towards ATP. This migration of HK is due to cross-diffusion, where enzymes aggregate near higher substrate concentrations. This is contrary to regular Fick diffusion, where enzymes diffuse from higher to lower concentrations.
Mannose caused lesser focusing of HK compared to D-glucose
indicating that catalysis may cause the focusing effect.
Compared to D-glucose, HK has a higher binding affinity for mannose. Additionally, mannose is turned over slowly by HK. In the experiment, mannose caused lesser focusing of HK compared to D-glucose. These results indicate that catalysis may cause the focusing effect. This hypothesis was confirmed by a competition assay between mannose and D-glucose. HK, ATP, and Mg2+ were introduced into the central channel, one flanking channel contained D-glucose, and the other contained mannose. A significant chemotactic shift was seen towards the D-glucose channel suggesting that catalysis, and not substrate binding, causes the enzyme movement.
The next question that arises is whether the enzymes can still display chemotactic behavior if only the initial substrate (glucose) is provided. To answer this, the enzyme cascade of HK, Iso, PFK, and Ald was set up and glucose was provided as the substrate. After glucose is processed by the first three enzymes, fructose 1,6-bisphosphate is produced. Therefore, the migration of Ald towards its substrate, fructose 1,6-bisphosphate was measured. The microfluidic device was designed as follows: D-glucose, HK, Iso, PFK, ATP, and Mg2+ in the right flanking channel, Ald in the central channel, and buffer in the left flanking channel. Ald moved into the channel where its substrate was being formed in situ indicating that the chemotaxis behavior still holds when the substrates are being channelled. Additional control experiments that lacked either Iso, PFK, or D-glucose did not show any discernable movement of Ald.
Chemotaxis behavior still holds when the
substrates are being channelled
It can be argued that the enzyme movement in buffer is not representative of the crowded cellular environment which might hinder the chemotactic movement. To test this possibility, 20% Ficoll PM 70 was used. Ficoll PM 70 is a highly branched polysaccharide that acts as a synthetic crowding agent. Although Ficoll slowed down the chemotaxis of enzymes, the co-localization of HK and Ald enzyme aggregates was still observed in a sealed hybridization chamber that had the same crowding conditions as the microfluidic experiments. However, this experiment could only track large enzyme aggregates. It could not precisely locate rapidly diffusing smaller enzyme aggregates, which are more representative of the intracellular environment.
Enzymes use chemotaxis to migrate to the substrate
as an alternative route in a
crowded and chaotic environment
Glycolytic enzymes need to process large volumes of substrates. To do so, they build a production line where the substrates are channeled from one enzyme to the next. However, if substrates are limiting, the enzymes need to find them to perform this laborious task. Therefore, enzymes use chemotaxis to migrate to the substrate as an alternative route in a crowded and chaotic environment. This study demonstrates two guiding forces behind the chemotactic movement: (1) the cross-diffusion of enzymes up the substrate gradients and (2) the dependence of cross-diffusion on catalysis when all the substrate requirements are met. The authors conclude that the direct interaction of enzymes is not required for substrate processing; only the presence of a substrate gradient causes the aggregation of enzymes.
About the work:
The article is based on recent paper published in Nature Chemistry, 2017; Substrate-driven chemotactic assembly in an enzyme cascade
Authors: Zhao X., Palacci H., Yadav V., Spiering M.M., Gilson M.K., Butler P.J., Hess H., Benkovic S.J., Sen A.
For publication, see: https://www.nature.com/articles/nchem.2905
Author:
Ananya Sen is currently a graduate student in Microbiology at the University of Illinois at Urbana-Champaign. When she’s not studying oxidative stress, she is busy pursuing her passion for scientific writing. Currently she contributes articles to ASM, ScienceSeeker, and her own blog where she discusses the history of various scientific processes. She is an ardent reader and will happily discuss anything from Jane Austen to Gillian Flynn. Her graduation goals include covering all the national parks in the U.S. with her sidekick Oscar, a Schnauzer/Pomeranian mix.
Editors: Rajamani Selvam and Arunima Singh
Rajamani Selvam is currently a Neuroscience Ph.D. student at University of Connecticut Health, Farmington, CT. Her research focuses on understanding the interactions between growth factors and endocannabinoids in modulating acute synaptic transmission in the brain. Post-graduation, she is interested in pursuing a career in medical communications. She is passionate about communicating STEM education and outreach to middle and high schoolers. She is also a mentor for 1000 girls 1000 futures program, New York Academy of Sciences. Away from science, she is an artist and enjoys leisure travel.
Arunima Singh obtained her Ph.D. from the University of Georgia and is currently a post-doc at the NYU. A computational structural biologist by training, she enjoys traveling, reading, and the process of mastering new cuisines in her spare time. Her motivation to move to New York was to be a part of this rich scientific, cultural, and social hub.
Illustrator
Ipsa Jain provided the cover image. She is a post-doctoral fellow at Instem, Bangalore. She tries to communicate science through visual arts as a medium. Collecting graphic books, tree trash, and reading brain pickings is few of her favourites. Follow and purchase her artwork at Ipsawonders (Facebook, Twitter, and Instagram). She will be happy to hear praises and non-praises at ipsajain.31@gmail.com.
Blog design: Rajamani Selvam
The contents of Club SciWri are the copyright of PhD Career Support Group for STEM PhDs {A US Non-Profit 501(c)3}. (PhDCSG is an initiative of the alumni of the Indian Institute of Science, Bangalore. The primary aim of this group is to build a NETWORK among scientists, engineers and entrepreneurs).
This work by Club SciWri is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
