A moving enzyme story

We now better understand the importance of movement for the functioning of enzymes, and how the structure of such proteins is critical for catalysis, thanks to research by scientists in the United States. It is hoped that an awareness of the significance of motion for the proper functioning of enzymes and other kinds of proteins will lead to new ways of designing more effective drugs that target enzymes.

 

Although it has long been known that enzymes exist in different shapes and forms, and that changes in shape allow them to bind to their substrates and co-factors, this is the first evidence that movement has a role in the actual chemistry of a reaction. The researchers, from the Scripps Research Institute and Pennsylvania State University, whose work on the E. Coli enzyme has been published in the journal Science [Bhabha et al. Science (2011) doi: 10.1126/science.1198542], showed that even small oscillations can have a substantial impact on how an enzyme functions.

 

Technological innovation has allowed the team, led by Peter Wright, to monitor protein motions lasting just milliseconds, the most relevant timescale to biology. After exploring the dynamics of the enzyme dihydrofolate reductase (DHFR), they realized that if these movements were prevented, and there was no alteration to the overall structure or other properties of the enzyme, then the enzyme is rendered defective for carrying out chemical reactions.

 

This led to an investigation of DHFR from the common bacterium Escherichia coli, using the enzyme as a model for examining how enzymes are able to catalyze chemical reactions. As DHFR is essential to bacterial cells, and human cells also use DHFR, it has been the target of many antibiotics and also been used in cancer chemotherapy. DHFR also encourages the conversion of the compound dihydrofolate (DHF) into tetrahydrofolate (THF), which is needed by cells for synthesizing DNA.

 

The DHFR uses a co-factor, NADPH, in its chemical reactions, and catalyzes the transfer of a negative hydrogen ion (hydride) from NADPH to DHF to produce THF. It is known that the loops around the active site are flexible, and one specific loop, the Met20, can adopt two different conformations during the catalytic cycle. To identify the importance of the oscillations, the scientists developed a mutation in the DHFR enzyme that stopped the Met20 loop from moving, and then checked the bacterial DHFR protein sequence against that of the human enzyme, to identify which amino acids they should change.

 

This technique allowed the team to successfully develop a rigidified mutant E. coli DHFR that was practically the same as the wild-type enzyme. However, NMR analysis showed that the Met20 loop and other parts of the active site were no longer flexible in the mutant. Also, the mutated E. coli enzyme transferred hydride much slower than that of the wild-type enzyme, resulting in a significant loss in enzyme function. The team was able to demonstrate that locking down the motion in the active site would prevent catalysis.

 

It is hoped it will be possible to harness the motions and integrate protein flexibility into the development of new enzyme-targeted drugs that either inhibit or increase enzyme function.