Probing Single-Molecule Enzymatic Dynamics of B-Glucosidase using Zero-Mode Waveguides

Abstract

β-glucosidases (BGLs, EC 3.2.1.21) are exo-type enzymes that hydrolyze β-glucosidic bonds from the non-reducing end of their substrates. BGLs are present in all living organisms (bacteria, archaea, and eukarya), and perform a range of functions. In bacteria and fungi, BGLs play an important role in cellulose saccharification, which catalyzes the hydrolysis of cellobiose and short cellooligosaccharide to glucose. Due to their potential biotechnological importance, a large number of BGLs from bacteria and fungi have been cloned and characterized. Interestingly, these enzymes often exhibit the enzymatic properties such as substrate and product inhibition. Thus, the dynamic molecular properties are too complicated to be fully understood by the use of conventional ensemble techniques. We then employed a single-molecule assay to probe the enzymatic dynamics of BGL.BGL1B from the wood-rotting basidiomycete Phanerochaete chrysosporium was used in this study. As the enzyme has a relatively low affinity for cellobiose (Km = ∼200 μM), µM concentration of fluorescent cellobiose is required to monitor the enzymatic reaction. Therefore, we employed a single-molecule assay using zero-mode waveguides (ZMWs). ZMWs comprise nanoscale holes in an aluminum film deposited on a fused silica coverslip, and can reduce the observation volume by more than three orders of magnitude relative to conventional microscopic techniques, allowing single-molecule observations at µM concentrations of fluorescent molecules in solution. Biotinylated BGL1B was immobilized in ZMWs through a biotin-streptavidin linkage. The BGL1B was immersed in a solution containing 1 μM tetramethlyrhodamine-conjugated cellobiose (TMR-cellobiose). The repeated appearance and disappearance of TMR fluorescence were often observed, indicating that immobilized BGL1B hydrolyzes TMR-cellobiose in ZMWs. Surprisingly, we have found that the enzymatic reaction is inhibited by glucose non-competitively at the lower concentration and competitively at the higher concentration.