Open any biology or chemistry textbook and entire chapters will be dedicated to detailing molecular processes crucial to life that are only made possible by seemingly magical proteins called enzymes. The magic is not fully understood, however, and each book will offer a somewhat different explanation for how enzymes work.
Now, Stanford chemists have peered inside a working enzyme and found that local electric fields focused at the active site might play a big role in helping it accelerate reactions.
The results are published in the current issue of Science.
Without enzymes, life wouldn’t be possible. Nearly every process in cells – DNA replication, protein synthesis, metabolism of food into energy and even steroid production – is made possible by an enzyme interacting specifically with its target substrate to transform it into something useful. Oftentimes, the reaction is so slow that it would take billions of years to occur without the enzyme’s involvement. Enzymes can accelerate these slow reactions by up to 25 orders of magnitude, and with great selectivity.
But when scientists have tried to design new enzymes, even by following the atomic blueprint of well-studied enzymes, things just don’t seem to click.
“Clearly it’s important to have the right pieces in place, but there seems to be something more,” said Steven Boxer, the Camille and Henry Dreyfus Professor of Chemistry at Stanford, and senior author on the new study. “There are a lot of really strong opinions about this, but one idea that’s emerged, mostly from simulations, is that electrostatic interactions within the enzyme might play an important role lowering the barrier for the reaction, but we haven’t had a way to measure this until now.”
One thing that scientists know happens when an enzyme is at work is that it binds the target molecule to its own “active site.” Here, the enzyme helps break and form bonds, transferring electrical charges around as it transforms the molecule in a step-by-step fashion.
This movement of charge interacts with electric fields from specific hydrogen bonds and other interactions between the enzyme and the molecule upon which it’s acting, called a substrate.
Some chemists have theorized that such electrostatic fields lower the barrier for the reaction as the substrate transitions to the final product, and that the better this connection, the more proficient the enzyme’s action.
Boxer applied a modern twist on a 100-year-old technique for measuring electric fields, and probed the active site of ketosteroid isomerase (KSI), an enzyme responsible for steroid metabolism. They found that the enzyme exerts an extremely large electric field on the substrate, and this would give a major boost to a molecule that would otherwise be sluggish to react.
Next, they repeated the experiment on versions of KSI featuring slightly altered active sites that were previously shown to be less effective at catalyzing the reaction. In comparing the natural KSI output to the mutants, the chemists found that the electrostatic field in the native enzyme was extremely large and responsible for as much as 70 percent of KSI’s performance.
“The reality is that it was never going to be all one thing or another, but 70 percent is a very significant contribution, and these experiments tell us that the electrostatic field is directly impacting the rate in this case,” Boxer said. “This shows that the electrostatic field lowers the barrier to reaction, and is really the key to catalysis in this enzyme.”
The effect is likely preserved across other enzyme systems, Boxer said, but its degree of contribution likely varies depending on the molecules involved. The new approach they have developed can be used to better quantify the electrostatic contribution to catalysis in other enzymes, so perhaps other chemists could use this information to make existing enzymes more efficient, or design novel enzymes or even chemical catalysts that amplify the electrostatic field.
“This work is basic science, and on its own is not going to solve the energy crisis or anything like that,” Boxer said. “But it will help us to better interpret a lot of good data that’s already out there, and in the broader sense this will help us understand what’s so unique about enzymes based on fundamental physical concepts.”
The study was co-authored by Stephen Fried and Sayan Bagchi, who conducted the work as a graduate student and a postdoctoral research fellow, respectively, at Stanford.
Steven Boxer, Chemistry: (650) 723-4482; firstname.lastname@example.org
Bjorn Carey, Stanford News Service: (650) 725-1944; email@example.com