Stochastic inhibitor release and binding from single-enzyme molecules

Abstract

Inhibition kinetics of single-␤-galactosidase molecules with the slow-binding inhibitor D-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultrasmall reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of ␤-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.␤-galactosidase ͉ enzyme kinetics ͉ fluorescence microscopy ͉ single molecule T he emergence of new assays for studying enzymes at the single-molecule level has profoundly extended our view of enzyme mechanisms. Whereas stochastic molecular behaviors are hidden by using bulk methods, single-molecule experiments have revealed that, in a population of enzymes, such as lactate dehydrogenase (1), phosphatase (2), or ␤-galactosidase (3), the catalytic rates of individual enzyme molecules are heterogeneous and do not interconvert quickly. Furthermore, it has been shown that the substrate turnover of individual ␤-galactosidase (4), cholesterol oxidase (5), or lipase (6) molecules undergoes dynamic fluctuations in sequential catalytic cycles. These two variations in enzyme activity are referred to as static and dynamic heterogeneity, respectively, and have both been attributed to different conformational states of the enzyme. Despite such heterogeneity, the Michaelis-Menten model derived from bulk enzyme experiments still holds for single-molecule experiments once a stochastic perspective is adopted (4,7,8).Traditionally, one of the most important tools to elucidate an enzyme's catalytic mechanism has been to study the enzyme in the presence of inhibitors. Here, we set out to correlate inhibition mechanisms established in bulk enzyme studies with singleenzyme molecule experiments. To date, inhibitors have been used in single-molecule studies to obtain information about conformational dynamics. Ha et al. (9) showed that it is possible to distinguish between free and inhibitor-bound states of a single-staphylococcal nuclease enzyme molecule. The binding of an inhibitor imposed a conformational constraint on the enzyme molecule resulting in a change of single-molecule polarization and intramolecular single-pair FRET. In this article, we report the direct observation of inhibitor release and binding from single-enzyme molecules by monitoring their substrate turnover.

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

“…The favorable spectroscopic properties of resorufin have been previously harnessed for the production of sensitive in vitro substrates for esterases and glycosidases (Tokutake et al, 1990;Beisson et al, 2000;Coleman et al, 2007), including single-molecule detection (English et al, 2006;Gorris et al, 2007). Indeed, earlier work has highlighted the utility of resorufin b-glycosides for imaging the activity of GHs, including exo-b-galactosidase in yeast (Saccharomyces cerevisiae; Wittrup and Bailey, 1988) and exo-b-glucosidase in animal cells (Hays et al, 1998).…”

Perturbation Waves in Proteins and Protein Networks: Applications of Percolation and Game Theories in Signaling and Drug Design

“…Evidence from single-molecule experiments on a tetrameric enzyme ascertained the textbook model of cooperativity at a single-molecule level showing that ligand binding of the four subunits was not independent from each other: the majority of tetramers showed a one-step jump from no activity to the highest state of activity without observable intermediate states. Moreover, released inhibitors left occasional conformational traces behind causing heterogeneity among protein molecules [59]. So, it is straightforward to presume that complex communication-patterns emerge both inside individual proteins and in their oligomeric complexes.…”

“…In the current review we restrict our summary to those, which can be interpreted as propagating conformational changes of proteins. The framework of perturbation waves can be applied to most processes above, and also allows the construction of efficient models to understand the propagation of noise, the fluctuations and diversity of individual cells governing phenotype variations, cellular movement, cell division and other physical rearrangements inside the cells and the threshold between smaller disturbances and larger damages [58,59]. Fig.…”

Single enzyme molecule assay with time resolution using capillary electrophoresis

“…Figure 10 shows the resultant electropherogram from a continuous flow assay of the individual β -galactosidase molecules as the inhibitor d -galactal dissociated (Craig et al 2012b ). In a similar study, although not using a CEbased methodology, Walt proposed that the dissociation of bound d -galactal from a given individual β -galactosidase molecule occurred simultaneously at all four subunits (Gorris et al 2007 ). No stepwise increase in activity as inhibitor molecules dissociated one at a time from a given enzyme molecule was noted.…”

“…In a later study, using fluorescence microscopy, Yeung found that catalytic rate varies over time for individual molecules of lactate dehydrogenase, but not for reactions catalyzed by single metal ions, suggesting that different conformational states might be a cause of the heterogeneity (Tan and Yeung 1997 ). Further studies, using CE (Craig et al 1996 and fluorescence microscopy (Gorris and Walt 2007), have consistently showed, using different enzymes, that catalytic rates vary between different individual molecules and over time for a given molecule. Such heterogeneity is not limited to catalytic rate.…”