Scientists have combined NMR and X-ray crystallographic techniques to demonstrate that the catalytic activity of an enzyme is linked with its ability to move. Focusing on the E.coli dihydrofolate reductase (DHFR) enzyme, which is a target for some antibiotics, collaborators at Scripps Research Institute and Pennsylvania State University demonstrated that if a normally flexible loop by the enzyme’s active site is prevented from oscillating, the catalytic activity of the enzyme is significantly reduced.
They suggest that taking enzyme motion into account when designing new drugs might improve their specificity or effectiveness. The results are published in Science in a paper titled “A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis.”
DHFR is found in both bacterial and human cells. Bacteria need the enzyme for survival, and in rapidly dividing human cells the enzyme is the target of some anticancer drugs such as methotrexate, the research team notes. Previous work at the laboratory of Scripps Research Institute professor Peter Wright, Ph.D., has shown that loops surrounding the active site of DHFR are flexible, and one of these, termed theMet20 loop, can take on two different conformations during the catalytic cycle.
To investigate the importance of these loops’ ability to move or oscillate, the research team synthesized a mutant form of bacterial DHFR in which the substitution of specific amino acids produced a Met20 loop that was far less flexible. Knowing which amino acids to change was helped by a comparison of bacterial DHFR with the human version of the enzyme, in which the Met20 loop is naturally more rigid.
When the resulting mutant bacterial enzyme was compared with the wild-type enzyme using X-ray crystallography, it appeared virtually identical. However, using NMR analysis the researchers found that the Met20 loop and other parts of the active site were no longer flexible. Significantly, this reduced mobility led to a 16-fold reduction in the enzyme’s normal activity, which is to transfer hydride from NADPH to dihydrofolate (DHF) in the production of tetrahydrofolate (THF).
“This is the first demonstration that motions play a role in the actual chemistry of a reaction,” claims Dr. Wright, who is chair of the department of molecular biology and member of the Skaggs Institute for Chemical Biology at Scripps Research.
The researchers suggest that the natural motility of the active site in bacterial DHFR acts to bring NADPH and DHF closer together and facilitate the transfer of hydride. “We think that mutations prevent the enzyme from clamping down on the hydride donor and acceptor so they can no longer get as close to each other as is necessary for efficient catalysis,” notes Scripps co-author Gira Bhabha, Ph.D.
The scientists hope that ultimately it may be possible to incorporate motility modulation into the design of drugs that either inhibit or increase enzyme function. In the case of DHFR, the difference between the natural motility of the bacterial and more rigid human enzyme could be a point for exploitation and potentially reduce the serious side effects of drugs that target DHFR, they suggest. “It’s a difficult and challenging problem, but it could have a huge impact,” Dr. Bhabha concludes.