  Enzymes have been shown to diffuse faster in the presence of their reactants.
Recently, we revealed new insights into this process of enhanced diffusion
using single-particle tracking (SPT) with total internal reflection
fluorescence (TIRF) microscopy. We found that the mobility of individual
enzymes was enhanced three fold in the presence of the substrate, and the
motion remained Brownian. In this work, we compare different experimental
designs, as well as different data analysis approaches, for studying single
enzyme diffusion. We first tether enzymes directly on supported lipid bilayers
(SLBs) to constrain the diffusion of enzymes to two dimensions. This
experimental design recovers the 3-fold enhancement in enzyme diffusion in the
presence of the substrate, as we observed before. We also simplify our system
by replacing the bulky polymers used in the prior chamber design with a
SLB-coated surface and glycerol. Using this newly-designed SLB/glycerol
chamber, we compare two different analysis approaches for SPT: the mean-squared
displacement (MSD) analysis and the jump-length analysis. We find that the MSD
analysis requires high viscosity and large particles to accurately report the
diffusion coefficient, while jump-length analysis depends less on the viscosity
or size. Furthermore, the SLB-glycerol chamber fails to reproduce the enhanced
diffusion of enzymes because glycerol inhibits enzyme activity.
