Be still, enzyme: NMR points to motion control

Enzymes in motion

"Ever since the first X-ray structures of proteins emerged, scientists have been talking about proteins as though their structures were fixed in space," says Peter Wright of Scripps Research. "But that is not how proteins work. They are like the machines we build. They have moving parts, and they need motion to work."

Wright and his teamed have now turned to the flexibility of NMR spectroscopy to study the enzyme dihydrofolate reductase (DHFR) from the common bacterium Escherichia coli. DHFR catalyses the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF), which is used in DNA synthesis. The bacterium cannot live without DHFR, making it a useful target for antibiotics. Human cells also use their own DHFR particularly when dividing, making the human equivalent of this enzyme a target for methotrexate, the anticancer drug. Previously, Wright and colleagues demonstrated that loops surrounding the active site of the enzyme are flexible and that one of the loops in particular, the Met20 loop adopts two different conformations during the catalytic cycle. But, the significance of the enzymic motions remained obscure, until now.

Wright, graduate student Gira Bhabha, and colleagues from both Scripps Research and Pennsylvania State University used NMR to visualize the motion of the enzyme in solution, capturing movements on a biologically relevant timescale, something that the static results from even the most powerful diffraction studies cannot do. To figure out how important enzyme oscillations are to functionality, the team created DHFR mutants that precluded Met20 loop motion; they discerned which amino acids to change on the basis of sequence comparisons with human DHFR the Met20 loop in which is more rigid.

They first used X-ray crystallography to obtain a structure of the mutant enzyme and to show that it was almost identical to the wild-type enzyme. They then looked at the dynamic NMR of the mutant and could see that its Met20 loop was no longer flexible. Moreover, the mutant enzyme was sixteen times slower at carrying out its main function, the hydride transfer reaction. Such a rate reduction represents a significant loss of function.

Locking down enzymes

One explanation as to why making the Met20 loop rigid should make the enzyme so inactive is that in normal activity, the E. coli DHFR oscillates and nudges its co-factor closer to the target substrate DHF. This allows hydride to transfer from NAPDH to DHF to generate THF more efficiently than occurs in the rigid mutant. Without the necessary jiggling of the active site, the molecules only rarely come into suitably close proximity for the chemical reaction to occur. "We think that the 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," explains Bhabha.

The diagram illustrates the Scripps finding that a mutant strain of the E. coli enzyme dihydrofolate reductase undergoes motions essential for efficient catalysis. The hydride transfer reaction is shown in the centre of the enzyme locus. Finding ways to limit such oscillations in enzymes associated with disease might lead to novel drugs to either inhibit or increase enzyme function. "The idea is to harness these motions in drug design," added Bhabha. "It's a difficult and challenging problem, but it could have huge impact."

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.