Can enzymes change the local pH value?

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Researchers at the Ruhr University Bochum have developed a new method to determine the pH value in a narrowly defined volume of liquid. So far, a reliable measurement with a pH meter has only been possible in larger and uniform volumes. The team from the Cluster of Excellence Ruhr Explores Solvation, Resolv for short, describes the new method, which is based on terahertz spectroscopy, in the journal Angewandte Chemie, published online in advance on November 6, 2020.

For the work, the teams from the Chair of Physical Chemistry II around Prof. Dr. Martina Havenith and from the Chair of Theoretical Chemistry led by Prof. Dr. Dominik Marx. "There is increasing evidence that biological reactions do not depend so much on the global chemical properties of a solution, but that the local conditions in the immediate vicinity of an enzyme are decisive," says Martina Havenith. These include, for example, the pH value or local charge states. "It is important for us not only to measure these local properties, but to be able to calculate them predictively - for example, if we want to make enzymes usable as biocatalysts and to create the optimal environmental conditions for them," says Dominik Marx.

Tests with the amino acid glycine

The scientists worked with a solution of the amino acid glycine. This has two functional groups that can accept or release protons. The acid can therefore be in different protonation states, which can be varied by changing the pH of the solution.

The chemists examined glycine solutions using terahertz (THz) spectroscopy. They send radiation in the THz range through the solution, which absorbs part of the radiation. They represent the absorption pattern in a certain frequency range in the form of a spectrum. At the same time, they also calculate the THz spectra of these aqueous solutions for different pH conditions.

Different spectra depending on the pH value

Depending on the protonation state of glycine, the spectra turned out to be very different. Using complex computer simulations, so-called ab initio molecular dynamics simulations, the group investigated why. With this method, the researchers can assign certain areas of a spectrum - the so-called bands - to the movements of different bonds in the molecule. They showed how the different protonation states were reflected in the spectrum. While deprotonated glycine (high pH value) causes almost no peaks in the terahertz spectrum, protonated glycine (low pH value) produces clearly visible bands. The spectrum of an intermediate state, the glycine zwitterion (neutral pH value), was in between. In this way, the researchers received, so to speak, a fingerprint of the protonation, measured as a function of the pH value. They showed that the intensity of the spectrum in the range between 0 and 15 terahertz correlates with the pH value.

In further experiments, the researchers demonstrated that the method also works for the amino acid valine and for small peptides. "In the future, this fundamental knowledge will open up new possibilities for non-invasive determination of local charge states on the surface of biomolecules," sums up Martina Havenith.

The work was funded by the Deutsche Forschungsgemeinschaft within the framework of the Resolv Cluster of Excellence (EXC 2033 - 390677874) and the project with the grant number MA 1547/11 as well as by the European Research Council in the project "THz-Calorimetry", grant number 695437.

Martina Havenith et al .: Probing local electrostatics of glycine in aqueous solution by THz spectroscopy, in: Angewandte Chemie International Edition, 2020, DOI: 10.1002 / anie.202014133

Prof. Dr. Martina Havenith
Physical Chemistry II
Faculty of Chemistry and Biochemistry
Ruhr-University Bochum
Tel .: +49 234 32 24249
Email: [email protected]

Prof. Dr. Dominik Marx
Chair for Theoretical Chemistry
Faculty of Chemistry and Biochemistry
Ruhr-University Bochum
Tel .: +49 234 32 28083
Email: [email protected]