We've had a rather static view of enzymes over the years, despite the wealth of information that has come from both classical enzymology and structural biology. However, enzymatic catalysis and internal protein dynamics are closely linked, so how do we begin to study the dynamics of catalysis, ask what internal motions occur, and decide whether these movements are related to catalytic action?

In Science, Kern and colleagues now describe studies using NMR to characterize enzyme dynamics during substrate turnover in the human enzyme cyclophilin A (CypA). The cyclophilin family of enzymes catalyse the cis/trans isomerization of X-proline peptide bonds, where 'X' is any amino acid.

Previous methods to study enzyme dynamics during catalysis have looked at global motions or the motions at a specific molecular site, but NMR can simultaneously investigate movements at many atomic sites. Kern and co-workers therefore studied the movements of 160 amide nitrogen nuclei in the protein backbone of CypA while it was actively catalysing isomerization.

Following substrate addition, the authors found that NMR relaxation parameters that are sensitive to very fast motions (on the pico- to nanosecond time scales) did not detect any major motional changes. However, for 10 of the 160 probe nuclei, they observed changes in the R2 relaxation parameter that is sensitive to motions on the micro- to millisecond time scales.

For nine of these 10 residues — most of which lie in the region of the substrate binding site — Kern and colleagues found that the changes in R2 are likely to be dominated by substrate binding and dissociation. However, the authors observed changes in R2 for arginine 55 that indicate a role for this residue in both binding and isomerization.

R55 is an essential catalytic residue, the side chain of which is hydrogen bonded to the substrate. The authors observed that the motion of the amide nitrogen of R55 is strongly correlated with the microscopic rates of substrate turnover, and concluded that this amide nitrogen is likely to be detecting motional changes that are linked to transition-state rearrangements in the protein and/or substrate. By combining their results with structural data, the authors were also able to predict a reaction trajectory for CypA.

Kern and co-workers have described an elegant approach for identifying dynamic 'hot spots' during catalysis, and have found that the time scales of enzyme dynamics match those of substrate turnover. Ultimately, side-chain dynamics will be required to obtain a more detailed motion picture of catalysis, but NMR relaxation measurements during catalysis promise to be invaluable for understanding both enzyme dynamics and their connection with catalysis.