Single Molecule Enzymology

Enzymes are biomolecules that catalyze biochemical reactions. Bulk kinetic studies, which are performed on large ensembles of molecules, provide information about the average catalytic rate, substrate specificity, etc., of the population, but such studies do not reveal the properties of individual enzyme molecules, which may vary considerably. In addition, since molecular processes are stochastic, individual catalytic steps are indistinguishable in ensemble assays. Single molecule studies of enzyme catalysis provide access to these details, which can provide insight into the underlying mechanisms. Single molecule studies highlight the differences in catalysis within a population of molecules (static heterogeneity) and also within an individual molecule over time (dynamic heterogeneity). Observing single enzyme molecules enables us to detect transient kinetic and dynamic events, which are impossible to observe in bulk enzyme studies.

In our lab we use single molecule isolation using microwell array technology to simultaneously trap thousands of single enzyme molecules along with a fluorogenic substrate in femtoliter-sized wells. By observing and analyzing the fluorescence signal over time, we are able to study the catalytic properties of individual enzyme molecules. Using this platform we aim to answer fundamental questions such as: what is the origin of static heterogeneities observed across a population of enzymes? What affects the extent of activity heterogeneity that can be observed? What is the behavior of enzyme molecules in the presence of effectors such as inhibitors? How do mutations affect the activity distributions?  These questions, and more, can be addressed by the microwell array platform.

Fundamental Studies of Enzymes

In our lab, we are using single molecule detection technology to perform fundamental studies of enzymes. Currently, we are attempting to understand the underlying basis for the activity distribution of individual enzymes as previously reported1. By introducing a heating element to the system described above, we are able to probe the correlations between activity, conformation, thermal stability, and temperature for individual enzymes. Presently, we are investigating the correlation between the activation energy and the reaction kinetics of single enzyme molecules. We are able to monitor the activity within a wide span of temperatures enabling us to precisely measure thermodynamic values for single β-galactosidase molecules.

We are also interested in studying the conformational changes between different enzyme activity states. By using short heating pulses we introduce enough energy needed to overcome activation barriers. The activities of thousands of individual enzyme molecules are recorded before and after the heating pulses. In doing so, we are essentially replicating the classic Anfinsen experiment2 in which enzymes are denatured and then renatured in situ. We observe enzyme activity for a few tens of seconds in order to get an accurate measure of the activity of several thousand enzyme molecules. The microwell array is then heated to a temperature that denatures all the enzyme molecules in the array. Upon cooling, the enzymes renature and regain their activity. Once activity has been restored, we can measure the rates of the individual enzymes. Studying the redistribution of activity in the enzyme population enables us to explore the origin of static heterogeneity.

Restriction Endonuclease Kinetics

Under optimal conditions, restriction endonucleases are capable of mediating remarkably specific DNA cleavage. It is this quality that makes restriction endonucleases an indispensible laboratory tool for genetic modification and manipulation. However, the mechanism by which restriction endonucleases effectively discriminate between their cognate site and other DNA sequences is not fully understood. Under certain conditions, many restriction endonucleases are known to display “star activity” – relaxed specificity resulting in cleavage of DNA at sequences that differ from their normal recognition sequence, but the mechanism by which specificity is relaxed is not fully understood. We are using fluorescence spectroscopy and total internal reflection fluorescence microscopy to study the activity of restriction endonucleases, both at the ensemble and single-molecule level. We have developed a fluorogenic DNA substrate that allows us to track the progress of restriction endonuclease-mediated cleavage at a specific DNA sequence. This approach allows us to observe the kinetics of DNA cleavage at the canonical recognition sequence for a given restriction endonuclease, as well as at any suspected non-canonical sequences under conditions that lead to star activity. We are also using TIRF microscopy to elucidate the mechanisms of atypical restriction endonucleases, such as those that are active as monomers rather than dimers.

Single Enzyme Molecule Inhibition

When we added the slow-binding inhibitor D-galactal to a solution containing β-galactosidase and its substrate in the microchamber array (Figure 1), we were able to observe inhibition kinetics at the single molecule level (PNAS, 2007). Inhibited and active states of β-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 (Figure 1).

Inhibition

Figure 1: Inhibitor release from single enzyme molecules in a pre-steady-state experiment. β-galactosidase was first saturated with the inhibitor D-galactal. The inhibitor was then diluted 1000-fold such that the inhibitor concentration was too low to rebind to the catalytic sites of the enzyme. The diluted solution was quickly enclosed in the array and sequential fluorescence images were taken every 30 s. (A) A sequence of these images recorded after closing the chambers (left panel), 1020 s (middle panel), and 1920 s (right panel) shows a delayed onset of substrate turnover that we attribute to stochastic inhibitor release events. (B) Trajectories of fluorescence increase in the indicated microchambers. An empty chamber shows a constant background (red curve). (C) Distribution of off-times with 2 min binning time. (D) The semi-logarithmic plot illustrates a first-order release of inhibitor (© PNAS, 2007).

Enzyme Fragment Complementation Studies

Enzyme fragment complementation (EFC) is a special case of protein complementation assays (PCA). EFC is a phenomenon where two complementary fragments of the enzyme, which are inactive when separated, result in a functional enzyme molecule when combined. EFC of β-galactosidase from E.coli is a well-known system and is the basis for the blue-white technique used for screening bacteria in microbiology studies. Specifically, the α-complementation reaction consists of the association of an inactive deletion mutant of β-galactosidase (acceptor) with a peptide corresponding to the deletion (donor). The deletion mutant is a dimeric form of β-galactosidase that lacks activity. The deleted peptide activates the interface between monomers and restores β-galactosidase activity. Using the optical fiber microwell array set up, we have the ability to observe individual active species formed during the complementation process without immobilizing any of the molecules, as is typically done for these types of studies. The technique enables us to study the kinetics of the individual active species, the pathways involved in complementation, and the stoichiometry of the complementation process.3-7

Catalytic Activity of Single Nanoparticles

Metal nanoparticles are important catalysts in a variety of industrial applications. However, their catalytic properties are not fully understood, as they are highly dependent on the details of particle structure, including shape, size, composition, and crystallinity. Using a method analogous to that described for single molecule enzyme activities1, we can explore the individual catalytic activities of a heterogeneous ensemble of nanoparticles. The system chosen for this study is the oxidation of Amplex Red by hydrogen peroxide to form the fluorescent product resorufin, catalyzed by gold nanoparticles. The catalytic activities of the individual nanoparticles, as well as the detailed kinetics of the reactions, are being measured and statistically analyzed. These catalytic properties will be correlated with the optical and structural properties of the nanoparticles, via optical spectroscopy and scanning electron microscopy, respectively.

nanoparticle catalysis

Figure 2: Illustration of the fluorogenic catalysis of Amplex Red to resorufin by gold nanoparticles contained in microwells on an optical microwell array.

References

1. D.M. Rissin, H.H. Gorris, D.R. Walt, JACS, 2008 130 (15), 5349-5353
2. C. B. Anfinsen, E. Haber, M. Sela, F. H. White, Proceedings of the National Academy of Sciences of the United States of America 1961, 47, 1309-&.
3. Jacobson R H et al, Nature, 1994, 369, 761-766
4. DeVries J K and Zubay G, Journal of Bacteriology, 1969, pp. 1419-1425
5. Ulmann A, Jacob F and Monod J, J Mol Bio. 1968, 32, 1-13
6. Langley K E et al, Proc. Nat. Acad. Sci. USA, 1975, Vol. 72, No. 4, pp. 1254-1257
7. Langley K E and Zabin I, Biochemistry., 1976, Vol 15 , No . 22