The use of single-molecule detection in the resolution of complex enzyme kinetics.

Abstract

The bacterial enzyme para-hydroxybenzoate hydroxylase (PHBH) catalyzes a multiple step reaction that requires precise control of solvent access to the catalytic site during the reaction cycle and strict differentiation between structurally similar substrates. Previous studies on PHBH have indicated conformational changes in the active site are essential for some steps in the reaction. However, detection of the hypothesized conformational changes in the substrate binding phase of the reaction has remained elusive because of the need to synchronize the reaction to distinguish the conformational species existing at equilibrium. Single molecule spectroscopy allows one to follow each individual molecule in time, thus eliminating the need to synchronize and offering a way of detecting these conformational changes at equilibrium. In this thesis we report single-molecule fluorescence studies of PHBH in the absence of substrate that support the hypothesis that a critical step in substrate binding is the movement of the isoalloxazine between an in conformation and a more exposed open conformation. The conformational switches are followed directly by changes in fluorescence intensity, confirmed by studies with the Y222A mutant form of PHBH which suggest that the exposed conformation is fluorescent while the in-conformation is quenched. We note that many of the single-molecule fluorescence trajectories reveal a conformational heterogeneity, with populations of the enzyme characterized by either fast or slow switching between the in- and open-conformations. Our data also allow us to construct a model in which one flavin in the dimer inhibits the motion of the other. This model is supported by single-molecule studies of PHBH in sucrose, an osmolyte that favors dimer formation by minimizing exposed protein surface area. Sucrose also minimizes flavin dissociation from the enzyme, which has possible applications for the extension of the available observation time in future single-molecule experiments. Finally, the conformational changes in the subsequent stage of the catalytic cycle were followed by observing the interaction of PHBH with three substrate analogues, each of which stabilizes a different conformational state of PHBH upon binding. Single-molecule fluorescence allows direct observation of the conformational switching upon substrate binding, which previously had been inferred through kinetic studies and crystallographic structures.