Defining the Structural Basis for Allosteric Product Release from E. coli Dihydrofolate Reductase Using NMR Relaxation Dispersion

The rate-determining step in the
catalytic cycle of E. coli dihydrofolate
reductase is tetrahydrofolate
(THF) product release, which can occur via an allosteric or an intrinsic
pathway. The allosteric pathway, which becomes accessible when the
reduced cofactor NADPH is bound, involves transient sampling of a
higher energy conformational state, greatly increasing the product
dissociation rate as compared to the intrinsic pathway that obtains
when NADPH is absent. Although the kinetics of this process are known,
the enzyme structure and the THF product conformation in the transiently
formed excited state remain elusive. Here, we use side-chain proton
NMR relaxation dispersion measurements, X-ray crystallography, and
structure-based chemical shift predictions to explore the structural
basis of allosteric product release. In the excited state of the E:THF:NADPH
product release complex, the reduced nicotinamide ring of the cofactor
transiently enters the active site where it displaces the pterin ring
of the THF product. The p-aminobenzoyl-l-glutamate tail of THF remains weakly bound in a widened binding
cleft. Thus, through transient entry of the nicotinamide ring into
the active site, the NADPH cofactor remodels the enzyme structure
and the conformation of the THF to form a weakly populated excited
state that is poised for rapid product release.