Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

There is immense difficulty in mapping out the complete details of an enzyme's mechanism, especially those that catalyze an acid-base reaction, owing to the simple fact that hydrogen atom positions are rarely known with any confidence. Ultrahigh-resolution X-ray and, better still, neutron crystallography can provide this crucial layer of information. We paired these techniques to reveal the catalytic mechanism of dihydrofolate reductase (DHFR), an enzyme necessary for nucleotide biosynthesis and a classical drug target. In a complex that closely resembles the catalytically active state, DHFR stabilizes a particular substrate conformer and likely elevates the pK_a of the substrate atom that is protonated. This protonation occurs directly via water, with its access to the substrate regulated by structural fluctuation of the enzyme.