Scripps researchers use mutant E. coli enzyme to enhance understanding of enzyme mechanisms

Slight oscillations lasting just milliseconds can have a huge impact on an enzyme’s function, a finding that not only enhances understanding of enzyme mechanisms but also opens the door for more specific and effective drug design

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LA JOLLA, Calif.—Scientists at the Scripps Research Institute have made an important discovery about the role that movement plays in the function of enzymes and proteins, a finding that not only has broad implications for our understanding of enzyme mechanisms, but for more specific and effective drug design as well.

In a study recently published in the journal Science, the Scripps team describes its use of a model of the enzyme dihydrofolate reductase (DHFR) from the usually harmless, but food poisoning-causing, bacterium Escherichia coli to observe how slight oscillations lasting just milliseconds have a huge impact on an enzyme's function. The scientists further observed that by blocking these movements, without changing the enzyme's overall structure or any of its other properties, the enzyme is rendered defective in carrying out chemical reactions.

As the senior author of the study, Peter Wright, points out, protein motions are critical for biological functions, but their precise role in enzyme catalysis remains unclear. Although there is convincing evidence that conformational fluctuations are essential for the mediation of substrate and cofactor binding as well as product release and can be rate-limiting for enzyme turnover, the importance of protein flexibility for progression along the chemical reaction coordinate remains a matter of debate.

"This is something we have been working on for a very long time," says Wright, chair of the Department of Molecular Biology and member of the Skaggs Institute for Chemical Biology at Scripps Research, "but technology has developed to the point now where one can get extremely detailed information on a millisecond timescale."

DHFR spurs the conversion of a compound called dihydrofolate (DHF) to a different form, tetrahydrofolate (THF), which is needed by cells for synthesis of DNA. In its chemical reaction, DHFR uses a helper or co-factor, called NADPH. It catalyzes the transfer of a hydride (a negative hydrogen ion) from NADPH to DHF to produce THF. Previous studies by Wright and others have shown that the loops surrounding the active site are flexible, and that one of the loops in particular, the Met20 loop, can adopt two different conformations during the catalytic cycle.

For this study, Wright and his colleagues produced a rigidified mutation in the DHFR enzyme, hypothesizing that this "dynamic knockout" mutant designed to impair the dynamics of the Met20 loop in DHFR would give direct information on the role of active-site loop fluctuations in catalysis by the E. coli enzyme.

The researchers then examined the DHFR enzyme using X-ray crystallography in combination with nuclear magnetic resonance (NMR) spectroscopy. Doing so allowed Wright's team to capture protein motions "in a timescale that is relevant to biology, from microseconds to milliseconds to seconds." Observing that the mutant enzyme's structure was almost identical to the wild-type enzyme, the scientists used NMR analysis to see that the Met20 loop and other parts of the active site were no longer flexible in the mutant. The enzyme transferred hydride at a rate that was 16 times slower than that of the wild-type enzyme, representing a substantial loss in enzyme function.

"This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond timescale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for attempts to design novel protein catalysts," the researchers concluded.

These findings represent a paradigm shift in the way we view protein structures, Wright says. Historically, scientists have viewed them as structures fixed in space, but that is not how they work, he says.

"They are like the machines we build," Wright explains. "They have moving parts, and they need motion to work. This is the first demonstration that motions play a role in the actual chemistry of a reaction."

In addition, taking motion into account when designing drugs to either inhibit or increase enzyme function could result in more effective or more specific drugs, Wright says. In this particular case, "it might help reduce the serious side effects of drugs that target DHFR," he says.

Next, Wright's team will work to expand their understanding of how enzymes work, and they are particularly interested in investigating disordered proteins, which Wright says represent a class of "ideal drug targets."

"This is all very much a grassroots effort at this point," he says.

The study, "A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis," was published in the April 8 issue of Science. The study was supported by the National Institutes of Health (NIH) and the Skaggs Institute of Chemical Biology. Wright's co-authors were Damian C. Ekiert, Ian A. Wilson and H. Jane Dyson at Scripps, and Jeeyeon Lee, Jongsik Gam and Stephen J. Benkovic at Pennsylvania State University.

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