Researchers have developed a tool that greatly accelerates enzyme research

Scientists now know that these transformations are driven by enzymes, which are protein molecules composed of amino acid chains, whose role is to accelerate or catalyze the transformation of one molecule (substrate) to another (product)

Polly Fordyce, assistant professor of bioengineering and genetics at Stanford University, said: "With the help of enzymes, a chemical reaction can occur within a few seconds, and its occurrence may be longer than the life cycle of the universe itself.

Although there is now a lot of understanding about enzymes, including their structure and the chemical groups they use to promote reactions, it is about how their form is connected to their function, and how they can achieve such amazing speed and specificity.

A new technology developed by Fortes and her colleagues at Stanford University may help change this situation, and the technology was published this week in the journal Science

By allowing scientists to explore in depth where substrate binding occurs outside of the enzyme’s small “active site”, HT-MEK can reveal clues about how even the most distant parts of the enzyme work together to achieve their extraordinary reactions Sexual

Fordyce said: "It's like we are holding a flashlight now, not only illuminating the active part, but illuminating the entire enzyme

Enzyme skills

HT-MEK is designed to replace the laborious process of purifying enzymes

However, this process is expensive and time-consuming, so just like the audience staring at the magician's hand when the magician performs a magic trick, the researchers mostly limit their scientific research to the active part of the enzyme

But any connoisseur of magic knows that the secret of successful magic lies not only in the movements of the magician's fingers, but also in the dexterous positioning of the arm or torso, the clattering that misleads the audience, or the discrete movements that occur under the stage and are invisible to the audience

"We eventually want to do the enzyme trick ourselves," Fordyce said

Enzyme experiments on the chip

HT-MEK combines two existing technologies to quickly speed up enzyme analysis

The second is the cell-free protein synthesis technology.

Fordyce explained: "We have achieved automation so that we can use the printer to deposit micro-views of the synthetic DNA encoding of the enzyme we want onto a glass slide, and then line up nanoliters filled with protein promoter mixtures at these spots.

Because each cell contains only one millionth of a liter of material, scientists can design thousands of variants of an enzyme in one device and study them in parallel

When the research team applied their technology to an enzyme called paa, they found that mutations outside the active site affected its ability to catalyze chemical reactions—in fact, most of the amino acids that make up this enzyme "Residues" have an effect

Scientists have also discovered that a surprising number of mutations cause paa to misfold into an alternative state that cannot be catalyzed

"This is one of thousands of enzymes," Herschlag emphasized
.
"We hope to have more discoveries and more surprises
.
"

Accelerated development

Researchers say that if widely adopted, HT-MEK can not only improve our basic understanding of enzyme functions, but also catalyze the advancement of medicine and industry
.
"Many of the industrial chemicals we use today are harmful to the environment and unsustainable
.
But enzymes work most effectively in water, the most environmentally friendly substance we have," co-first author Daniel Moher of the study Tari (Daniel Mokhtari) said that he is a Stanford graduate student at Hirschlag and Ford Labs
.

HT-MEK can also accelerate a drug development method called allosteric targeting, which aims to increase the specificity of drugs by targeting sites other than the active site of the enzyme
.
Due to the key role that enzymes play in biological processes, it is a commonly used drug target
.
But some are considered "unavailable drugs" because they belong to a family of related enzymes and have the same or very similar active sites.
Targeting them may cause side effects
.
The idea behind allosteric targeting is to create a drug that can bind to certain parts of enzymes, such as their surface, but still control certain aspects of catalysis
.
"With paa, we see the functional connection between the surface and the active site, which gives us hope that other enzymes will have similar goals," Markin said
.
"If we can determine where the allosteric targets are, then we can start the more difficult task of designing drugs for them
.
"

The large amount of data that HT-MEK is expected to generate will also be of great benefit to computational methods and machine learning algorithms, such as the AlphaFold project funded by Google, which aims to deduce the complex 3D shape of the enzyme only from the amino acid sequence of the enzyme
.
Mokhtari said: "If machine learning has any chance to accurately predict the function of an enzyme, it will need the kind of data provided by HT-MEK for training
.
"

In the future, HT-MEK may even allow scientists to reverse engineer enzymes and design their own customized varieties
.
"Plastic is a good example," Fordyce said
.
"We really want to invent an enzyme that can degrade plastic into non-toxic and harmless fragments
.
If the only important part of an enzyme is its active site, which is true, then we can do it, and it has Doing more
.
Many people have tried and failed
.
People think that one reason we can’t succeed is that the rest of the enzyme is very important to get the active site in the right shape and swing in the right way
.
"

Herschlag hopes that HT-MEK can be quickly applied to scientists
.
He said: "If you are an enzymologist trying to understand a new enzyme, you have the opportunity to observe 5 to 10 mutations in six months, or 100 to 1000 mutations in your enzyme in the same period.
, Which one would you choose?" "For the entire community, this is a tool that has the potential to replace traditional methods
.
"

Journal Reference :

1. CJ Markin, DA Mokhtari, F.
   Sunden, MJ Appel, E.
   Akiva, SA Longwell, C.
   Sabatti, D.
   Herschlag, PM Fordyce.
   Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics .
   Science , 2021; 373 (6553 ): eabf8761 DOI: 10.
   1126/science.
   abf8761