Single Molecule Study of Dihydroorotate Dehydrogenase (DHOD)

Figure. a) Fluorescence image of single DHOD molecules immobilized in agarose gel and fluorescence signal of a single DHOD molecule catalyzing substrate turnovers over time. The high and low fluorescence states correspond to the enzyme molecule in the oxidized and reduced state, respectively. Turnovers of single enzyme molecules were followed through the characteristic on-off fluorescence signal, which corresponds to the enzyme interconverting between the oxidized and reduced states during turnover.

By following the fluorescence of individual molecules immobilized in agarose pores, we are able to study the kinetics of enzyme catalysis, one molecule at a time. Compared to conventional ensemble studies, which observe the sum of unsynchronized reactions of a large ensemble of molecules and thus obtain ensemble-averaged information, single-molecule studies follow the reaction of one molecule and therefore obtain information of individual properties and their dynamic evolution in time. With the single-molecule approach, we had studied the kinetics of a flavoenzyme, Dihydroorotate Dehydrogenase (DHOD), and characterized a static heterogeneity in the activity of a Tyr318Leu DHOD mutant, with some molecules reacting 5-fold faster than others. Moreover, as sequential turnovers of the same molecule were followed, possible existence of dynamic disorder, in which the catalytic rate is fluctuating in time instead of being constant, was also investigated with our single molecule measurements. We showed that any dynamic disorder in the activity of the mutant DHOD, which probably can be attributed to conformational fluctuations, did not exist at the same time scale as the enzymatic turnover. Besides static and dynamic heterogeneity, our exploration of enzyme kinetics and mechanism at the single molecule level also includes investigating subunit activity of oligomeric proteins and identifying reaction intermediates that are hidden in ensemble-averaged studies, which are all beyond the scope of conventional ensemble approaches.

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