  The diffusion of enzymes is of fundamental importance for many biochemical
processes. Enhanced or directed enzyme diffusion can alter the accessibility of
substrates and the organization of enzymes within cells. Several studies based
on fluorescence correlation spectroscopy (FCS) report enhanced diffusion of
enzymes upon interaction with their substrate or inhibitor. In this context,
major importance is given to the enzyme fructose-bisphosphate aldolase, for
which enhanced diffusion has been reported even though the catalysed reaction
is endothermic. Additionally, enhanced diffusion of tracer particles
surrounding the active aldolase enzymes has been reported. These studies
suggest that active enzymes can act as chemical motors that self-propel and
give rise to enhanced diffusion. However, fluorescence studies of enzymes can,
despite several advantages, suffer from artefacts. Here we show that the
absolute diffusion coefficients of active enzyme solutions can be determined
with Pulsed Field Gradient Nuclear Magnetic Resonance (PFG-NMR). The advantage
of PFG-NMR is that the motion of the molecule of interest is directly observed
in its native state without the need for any labelling. Further, PFG-NMR is
model-free and thus yields absolute diffusion constants. Our PFG-NMR
experiments of solutions containing active fructose bisphosphate aldolase from
rabbit muscle do not show any diffusion enhancement for the active enzymes nor
the surrounding molecules. Additionally, we do not observe any diffusion
enhancement of aldolase in the presence of its inhibitor pyrophosphate.
