Map electric fields to help understand how enzymes work

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Graphic abstract. Credit: natural chemistry (2022). DOI: 10.1038/s41557-022-00937-w

Every moment, in the cells of our body, countless vital activities take place thanks to enzymes. These special proteins act as catalysts by speeding up the rate and improving the selectivity of chemical reactions without themselves undergoing permanent changes. Beyond their indispensable role in biology, enzymes are also essential for a myriad of processes in the food, pharmaceutical, agricultural and cosmetic industries.

Contrary to their ubiquity and importance, enzymes are poorly understood. In particular, scientists want to know what makes active sites of enzymes – the pocket-like region where accelerated chemical reactions occur – so powerful. While the three-dimensional atomic structures of the active sites of many enzymes have been visualized and mapped, the “invisible” structure of the electric fields inside an active site is mostly unknown. These electric fields are theorized to play an important role in forming a precise environment in active sites where molecules react and quickly transition to new molecules.

Now, a study co-led by Stanford researchers Chu Zheng and Yuezhi Mao has launched a new probe to measure and visualize electric fields inside an enzyme’s active site. The article, recently published in the journal natural chemistry, accounts for the orientation of electric fields at the reaction site and could help researchers calculate key chemical interactions at active sites. This knowledge, in turn, could lead to the creation of custom-made synthetic enzymes for industry, as well as greatly advancing the discovery and design of new drugs that interfere with or modulate the function of targets. enzymes.

“We have developed a new probe that can give us important insights into how electric fields are uniquely oriented in enzymes, which we believe is fundamental to the incredible catalytic power of enzymes,” said Zheng, graduate student in Steven G. Boxer’s lab, Camille Dreyfus professor of chemistry.

“At the basic level, we are trying to better understand how enzymes work, and in this study we add a new dimension by introducing electric field orientations that are believed to critically impact the catalytic functions of the enzyme,” said Mao, a postdoctoral chemistry researcher who works in the lab of Thomas Markland, an associate professor of chemistry at Stanford and also co-senior author.

A powerful new tool

Stanford’s Boxer lab pioneered the concept of interpreting enzyme functionality by measuring electrostatic interactions, which are present in all forms of matter and are specifically organized in three dimensions in large biological molecules.

“The origin of the amazing functionality of enzymes is a general question, and it applies not only to biological catalysis, but also to chemical catalysis, which is a huge undertaking,” Boxer said. “About 80% of all chemicals are made using catalysts, but what is actually responsible for reducing the free energy of activation? [to make the reaction occur faster] is not well understood for most reactions. Investigating the role of electric fields in enzyme function is central to our work,” said Boxer, chair of the chemistry department at Stanford’s School of Humanities and Sciences and co-lead author of the study.

The probe developed by the Stanford team is based on a technique, also developed in the Boxer laboratory, called Stark effect vibrational spectroscopy. This technique measures vibrational frequencies in probe molecules based on the wavelength of infrared light absorbed by its chemical bonds. Changes in these vibrational frequencies reveal information about the electric fields present. In this study, researchers investigated changes in the vibrational frequencies of chemical bonds in a probe made from a molecule called N-cyclohexylformamide. This molecule acts as an inhibitor, binding to the active site of an enzyme called alcohol dehydrogenase in the liver.

To visualize the electric field in the active site of liver alcohol dehydrogenase, the researchers targeted two bonds in the N-cyclohexylformamide probe about 120 degrees apart. This specific angle between the two bonds allowed the researchers to assess not only the strength or magnitude of the electric field, but also the orientation of the field. Previous Boxer lab studies of other enzyme active sites had reported on the magnitude of the electric fields but not their directions.

“We call this tool a bidirectional probe because with this probe we can measure the electric field in an active site in two different directions,” Zheng said. “By using the probe in this way, we can reconstruct and extract orientation information about the electric field. This has not been done in the past.”

Collecting this key metric first required chemical sleight of hand. One of the chemical bonds of the N-cyclohexylformamide probe – between a carbon atom and a hydrogen atom – is notoriously difficult to observe in protein environments. So the researchers replaced the hydrogen atom with the element’s heavier cousin, called deuterium. The new carbon-deuterium bond proved measurable and helped the researchers reveal the orientation of the electric field.

A precise enzymatic environment

The Stanford researchers combined their experimental data with computer simulations and quantum mechanical calculations to describe electric field interactions with deuterium-modified N-cyclohexylformamide at the active site of liver alcohol dehydrogenase. These properties were then compared to the electric fields present in water, acetone and other common solvents.

Notably, the researchers found that the orientation of the electric field in the active site of liver alcohol dehydrogenase differs significantly from the orientation of the electric field in the solvents they studied. This result supports the idea that enzyme active sites exhibit what scientists call a preorganized electrostatic environment, or an environment in which the precise positioning of amino acids and the electrostatic environment they create help reduce the energy required to a chemical reaction takes place. This could be the key to enzymes’ remarkable ability to catalyze reactions.

“With this study, we are helping advance the concept of correlating enzyme performance with both the magnitude and orientation of electric fields in active sites,” Mao said. “What we found is evidence that the electric fields in the active sites of enzymes are preorganized, and it’s an important clue to solving the mystery of why enzymes have their incredible abilities.”

The probe developed by the Stanford researchers could be used to study the active sites of many other enzymes. Expanding knowledge in this way will bring scientists and engineers closer to the ability to custom-design enzymes with spectacular new characteristics.

“The ultimate goal of this research is to allow us to design enzymes that have superb catalytic performance for biomedical and industrial applications,” Zheng said. “We’re still a long way off, but we’re making progress and understanding how enzymes work better than before.”

The evolution of an enzyme from changing electric fields and antibiotic resistance

More information:
Chu Zheng et al, Bidirectional vibrational probe reveals different orientations of electric field in solution and enzymatic active site, natural chemistry (2022). DOI: 10.1038/s41557-022-00937-w

Provided by Stanford University

Quote: Mapping Electric Fields to Help Unravel How Enzymes Work (2022, July 28) Retrieved July 28, 2022 from

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Maria D. Ervin