Structure determination of unstable reaction intermediates
Many
(bio)chemical reactions are complex, often involving several reaction
intermediates. For a thorough understanding of the reaction mechanism
it is necessary to know all steps occurring along the reaction
coordinate the kinetics of their interconversion, their equilibrium
constants, and the three-dimensional structures of the
intermediates. Crystallography, NMR, and electron microscopy are
excellent tools for determining three-dimensional structures to
atomic resolution, but are generally regarded as static methods,
averaging over space and data collection time.
Because data acquisition is generally lengthy and intermediates
are usually short-lived, their structures cannot normally be
determined by conventional approaches. Thus, transition state or
substrate analogues, inhibitors and mutants are traditionally used
to obtain mechanistic information. In the last two decades, however,
crystallographic structure determination of species that are
short-lived has become feasible either on ultra-fast time scales
("time-resolved crystallography") or by slowing reactions via
manipulation of, for example, temperature, pH, or slow substrates
("kinetic crystallography", "trapping"). Both approaches require
rapid and efficient initiation of the reaction in the crystal. We
have developed protocols for handling caged compounds in
crystallographic studies which opened up investigations of enzymatic
systems operating on phosphate-containing substances.