Neutron Scattering Reveals Enzyme Secrets

In just two neutron experiments, scientists have uncovered amazing information about the role of an enzyme that could assist in the development of drugs for aggressive cancers.

Utilizing neutron scattering at the Spallation Neutron Source and the High Flux Isotope Reactor, the scientists at the Department of Energy’s Oak Ridge National Laboratory were able to pinpoint the precise atomic-scale chemistry in serine hydroxymethyltransferase, or SHMT, a metabolic enzyme required for cell division.

In cancer, cells multiply quickly by taking over chemical reactions in the metabolic pathway that involves SHMT and other essential enzymes and turning the whole thing into a runaway train.

Cancer’s attempts to overtake it may be thwarted by creating an inhibitor that blocks the enzyme's function, which occurs early in the metabolic pathway. The team's results were published in Chemical Science by the Royal Society of Chemistry.

I think neutrons will be highly sought-after in future structure-based drug design. This paper is a good example of how quickly neutrons can produce information that has been the subject of debate for a very long time. Studies on SHMT function and its catalytic mechanism date back to the early 1980s.”

Victoria Drago, Study Lead Author and Biochemist, Oak Ridge National Laboratory

Decades of debate have centered on the precise catalytic mechanism and the functions of different amino acid residues in the active site of the enzyme. In the current study, the researchers found that this enzyme's ability to control chemical reactions is mediated by a single amino acid residue: glutamate.

The neutron data clearly show that the glutamate, which is an acid, has the proton on it. You might expect it to already have given up its proton. But because it is able to carry that proton around, it can transfer it back and forth. So, it acts as an acid and a base.”

Robert Phillips, Study Co-Author and Professor, University of Georgia

Within a cell’s mitochondria or energy producer, this enzyme functions in a process called one-carbon metabolism. Through the transfer of a carbon atom to tetrahydrofolate, a reduced form of folic acid, it transforms the amino acid serine into another amino acid known as glycine.

This reaction generates building blocks for the synthesis of nucleic acids (DNA and RNA), as well as other biological molecules required for cell division. Glutamate regulates this process.

In a previous experiment, the team used two techniques, neutron and X-ray crystallography at physiologically relevant room temperature, to better understand SHMT and map its protein structure before it interacts with tetrahydrofolate. In the current experiment, the researchers captured the enzyme in the next step, providing certainty about how the enzyme's reaction mechanism works.

Painting the Picture with Neutrons

Neutrons see lighter elements like hydrogen, whereas X-rays see heavier elements like carbon, nitrogen, and oxygen. Neutron diffraction at SNS and HFIR, in-house X-ray diffraction at ORNL, and synchrotron X-ray diffraction at Argonne National Laboratory's Advanced Photon Source all provided the team with the information required to definitively characterize the enzyme's chemical reaction.

Drago added, “Neutrons allow us to see hydrogen atoms and hydrogen drives chemistry. Enzymes are about 50% made up of hydrogen atoms. In terms of electrostatics, hydrogen also carries a positive charge, which dictates the environment of the enzyme. Once you have a crystal that will diffract neutrons, you have everything you need. You see the positions where hydrogens are located and, equally as important, the positions lacking hydrogens. You get the whole picture.”

As depicted in the animation, cancer cell mitochondria overproduce the SHMT enzyme, a tetramer made up of four identical peptide chains, or protomers, which are shown in gray. SHMT works by using pyridoxal-5′-phosphate, which is covalently bound to SHMT, and tetrahydrofolate, which are shown in gold and purple, respectively. Tetrahydrofolate functions as a substrate, binding to the active sites of all four protomers.

The hydrogen atoms, which flashed green, revealed the precise catalytic mechanism and the roles of various amino acid residues in the enzyme's active sites. Once the enzyme releases tetrahydrofolate, an inhibitor, shown in blue, could be designed to prevent further chemical reactions at these sites, effectively stopping the one-carbon metabolic pathway in cancer cells.

The locations of the hydrogen atoms determine protonation states of specific chemical groups inside the enzyme’s active sites. Thus, they provide information on the electric charge distribution or electrostatics. This knowledge is crucial to designing small-molecule inhibitors that would bind to SHMT, replacing tetrahydrofolate and halting the enzyme function.”

Andrii Y Kovalevskyi, Distinguished R&D Scientist, Oak Ridge National Laboratory

Cells contain thousands of enzymes that act as catalysts, speeding up biochemical reactions required for bodily functions such as breathing, hormone production, and nerve function. Enzymes also provide a place to store chemicals that target specific processes.

Methotrexate and fluorouracil are two well-known cancer drugs that target enzymes in the one-carbon metabolic pathway. However, SHMT occurs earlier in this pathway, providing an opportunity to stop cancer earlier.

However, the difficulties in treating cancer stem in part from its stealthy attacks on metabolic processes. Unlike drug resistance in infectious diseases, when one path fails, cancer adjusts other metabolic processes to overproduce cancer cells.

Kovalevsky added, “Now that we know the atomic details for SHMT, we can inform the design of an inhibitor to target this specific protein as part of a combination therapy. If you compare it to treating infectious diseases, this is much more difficult because in cancer chemotherapy, you usually target your own proteins, which is why patients experience side effects. In infectious diseases, the proteins you target belong to the viruses or the bacteria. But with cancer, you have to kill your own cells. The idea here is to kill the cancer sooner and have less of an effect on the patient.”

Speeding the Pace of Discovery

The team conducted its research using neutrons from the MaNDi instrument at SNS and the IMAGINE instrument at HFIR. ORNL's recent Proton Power Upgrade project upgraded all of SNS's instruments to provide stronger beams. Stronger proton beams result in more neutrons. More neutrons mean faster data collection with smaller samples, which helps scientists design smarter drugs to treat diseases.

William Nelson, director of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, stated, “Discovery research is absolutely essential. We’re moving ever closer to the space where, with the help of AI, we will be able to sequence a gene in somebody’s cancer, predict what the protein structure would look like and make a drug to tuck in; it will work great, and we’ll do it in an hour and a half. But we’re not there yet. So, the more we know about the actual protein structure, chemical structure and the way things interact, the better we’re going to be able train AI models to predict things we don’t know right away.”

Video Credit: Phoenix Pleasant/ORNL, U.S. Dept. of Energy