Enzyme activity studied under new lens!

Shenade Annie Kerketta, Amity University Kolkata

The knowledge that we have about how an enzyme works is through studying its ACTIVE SITE. New technology has been developed that gives us information about an enzyme by studying beyond the active site. The new technology is called HIGH THROUGHPUT MICROFLUIDIC ENZYME KINETICS (HT-MEK). This technology combines two processes which this article will describe.

What are enzymes?

Enzymes are biomolecules that alter the rate of reaction occurring inside an organism. They are, termed BIOCATALYSTS because they speed up the reaction inside a living organism. Without them, no metabolic activity would take place because the reactions would slow down considerably. They act as fuel for the body. Majority of them are made up of amino acids.

The mechanism behind enzyme activity:

ENZYME + SUBSTRATE → ENZYME-SUBSTRATE COMPLEX → ENZYME PRODUCT COMPLEX→ DESIRED PRODUCT + FREE ENZYME.

Here, the substrate depicts the substance on which the enzyme is acting. An enzyme has a specific structure that is essential for its working. An enzyme’s function depends upon its structure. If the structure changes, so do the function. The sequence of amino acids determines an enzyme’s structure. The site of an enzyme that binds to the substrate is called BINDING SITE. The site, which carries out the catalytic function is called CATALYTIC SITE. The binding site and catalytic site together form the ACTIVE SITE.

The active site is where the substrate binds. It contains residues that hold onto the substrate until the reaction is over. The active site brings about changes in the substrate, which leads to the formation of the product. To date, only the active site, has been explored. But, is it only the active site contributing to the enzyme activity?

The new technology – HT-MEK:

 HT-MEK is the successful result of the many trials conducted, to study parts of an enzyme beyond the active site. Two concepts are behind this new development.

1. Microfluidics: large spaces are reduced to small ones to carry out fluidic experiments. It is similar to the mechanism of integrated circuits.
2. Cell-free protein synthesis: here, only the parts needed to generate a protein are extracted and put together into a soupy extract. This soupy extract put into small wells formed (adjacent to each other) through microfluidics will be used to generate an ENZYME.

By combining, the two technologies, many variants of the same enzyme can be generated and studied side by side. Its function is analyzed by producing mutations in it. HT-MEK also helps to analyze regions other than the active site, whether other parts contribute to the enzymatic activity.

HT-MEK was implemented on PafA, an enzyme that is well studied. Researchers did find evidence to support that there are regions beyond the active site which contribute to catalysis. They also found that many of the applied mutations resulted in misfolds. The misfolds lead to an alternate structure, which hampered catalytic function, proving that structure is crucial to enzyme activity.

Conclusion:

There are many advantages of HT-MEK. Further studies, must be initiated and, must be accepted widely. HT-MEK could provide us with a whole new set of information concerning an enzyme’s mechanism. It could help with ALLOSTERIC TARGETING, a technique to increase drug specificity. It can be used to reverse engineer enzymes to generate new enzymes. Like, we could produce enzymes that could degrade plastics using HT-MEK. This new development could change our understanding of an enzyme so, further studies must be encouraged.

Also read: Extreme heat increases tree mortality in the sub-alpine forest

References: Markin, C. J., et al. “Revealing Enzyme Functional Architecture via High-Throughput Microfluidic Enzyme Kinetics.” Science, vol. 373, no. 6553, July 2021. science.sciencemag.org, doi:10.1126/science.abf8761. https://science.sciencemag.org/content/373/6553/eabf8761

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