Research Article

Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis

Jue Shi, Joe Dertouzos, Ari Gafni, Duncan Steel, and Bruce A. Palfey
PNAS April 11, 2006 103 (15) 5775-5780; https://doi.org/10.1073/pnas.0510482103

  1. Edited by Gordon G. Hammes, Duke University Medical Center, Durham, NC, and approved January 25, 2006 (received for review December 5, 2005)

Abstract

Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis, were characterized by employing single-molecule spectroscopy to follow the turnover kinetics of individual DHODA molecules, eliminating ensemble averaging. Because the enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, a single DHODA molecule exhibits stepwise fluorescence changes during turnover, providing a signal to determine reaction kinetics and study cooperativity. Our results showed significant heterogeneity in the catalytic behaviors of individual dimer molecules, with only 40% interconverting between the three possible redox states: the fully fluorescent (both subunits oxidized), the half-fluorescent (one subunit oxidized and the other reduced), and the nonfluorescent (both subunits reduced). Forty percent of the single dimer traces showed turnovers between only the fully fluorescent and half-fluorescent states. The remaining 20% of the molecules interconverted only between the half-fluorescent state and the nonfluorescent state. Kinetic analysis revealed very similar reaction rates in both the reductive and oxidative half-reactions for different DHODA dimers. Our single-molecule data provide strong evidence for half-sites reactivity, in which only one subunit reacts at a time. The present study presents an effective way to explore the subunit catalytic activity and cooperativity of oligomeric enzymes by virtue of single-molecule fluorescence.

The structural complexity and flexibility of proteins allow them to behave cooperatively, which requires conformational changes and intra- or intersubunit interactions (1, 2). Different degrees of cooperativity have been reported in a wide spectrum of biological processes (3–7). Half-sites reactivity reflects extreme negative cooperativity, where the activity of one site completely inhibits the reaction of the other site. In this article, we use a single-molecule approach to study catalytic cooperativity, and we present signatures of this behavior in single-molecule fluorescence trajectories. Analysis of the catalytic turnover of single molecules of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA), revealed not only half-sites reactivity but also significant catalytic heterogeneity.

Single-molecule spectroscopy follows enzymatic catalysis from a unique viewpoint and offers advantages for studying cooperative interactions. Steady-state kinetics reports on the catalytic cycle as a whole but is insensitive to processes that are not rate-limiting. Transient kinetics provides detailed information on some individual reaction steps, but cannot observe others for kinetic reasons and does not discriminate (without additional information) between catalytic and noncatalytic processes such as side-reactions. Single-molecule spectroscopy combines some of the best features of steady-state and transient kinetics, enabling individual reaction steps to be observed during steady-state turnover. Moreover, the ability to resolve microscopic catalytic states during turnover and directly observe their conversion to other microscopic states provides unique information that is unavailable after averaging a macroscopic ensemble, including the possibility of distinguishing the kinetics of reactions of individual subunits in cooperative enzymes.

Class 1A DHODAs (EC 1.3.3.1) catalyze the oxidation of (S)-dihydroorotate (DHO) to orotate (the only redox reaction in the biosynthesis of pyrimidine nucleotides) in some Gram-positive bacteria, anaerobic yeasts, and some protozoan parasites. DHODA from Lactococcus lactis is a homodimer (two 34-kDa subunits) with one flavin mononucleotide (FMN) bound to each subunit as a prosthetic group. During turnover FMN is reduced fully to FMNH2 when a hydride equivalent and a proton are abstracted from DHO. The flavin is then reoxidized by fumarate, the physiological electron acceptor. The FMN bound in the two subunits thus interconverts between the oxidized and reduced states. Similar to free FMN, DHODA-bound FMN is fluorescent in the oxidized state but not in the reduced state.

The fluorescence difference between the oxidized and reduced flavin makes it possible to study DHODA catalysis at the single-molecule level by using a confocal microscope. Steady-state kinetic studies of DHODA from L. lactis show that the enzyme has to be in the dimeric form to be catalytically active (8). Therefore, the signature fluorescence signal of a single DHODA dimer during turnover changes in a stepwise fashion, interconverting between the fully fluorescent, half-fluorescent, and nonfluorescent states, which correspond to the fully oxidized (FMN in both subunits), half-oxidized (FMN in one subunit and FMNH2 in the other subunit), and fully reduced (FMNH2 in both subunits), respectively.

Our single-molecule studies of DHODA demonstrate the existence of significant heterogeneity, manifested by three distinct catalytic behaviors. In the first, the dimers interconvert between all three fluorescent states; in the second, dimers interconvert only between the fully fluorescent and half-fluorescent states; and in the third, dimers interconvert between only the half-fluorescent and nonfluorescent states. Rates for reduction were observed to be similar among all three groups of DHODA dimers and all molecules also exhibited similar rates for oxidation. The results are interpreted in terms of half-sites reactivity.

Results

Trajectories of Single DHODA Molecules.

In the absence of substrates, the fluorescence of single DHODA dimers dropped to the background level in two steps, corresponding to the loss of FMN from each subunit (Fig. 1). The fluorescence loss was not due to photobleaching, because the length of the fluorescence time did not depend on the laser power. The duration of fluorescence was usually less than 5 s. Within the 20-s data collection time, recovery of fluorescence was only occasionally observed. The rate of FMN association would be <0.01 s−1 at subnanomolar concentrations of FMN, assuming diffusion-limited binding, which agrees with our observation that FMN reassociation is a rare event (<0.05 s−1).

Fig. 1.
Fig. 1.

Fluorescence trajectory of a single DHODA molecule. The fluorescence intensity drops in two steps, probably as the result of FMN dissociation from each subunit of DHODA.

Heterogeneity in Turnover.

When single enzyme molecules were observed in the presence of substrates, the flavin fluorescence switched between high, intermediate, and nonfluorescent levels, suggesting that the dimeric enzyme was turning over between the three possible redox states (fully oxidized, half-oxidized/half-reduced, and fully reduced). In principle, FMN dissociation from the holoenzyme could have caused the loss of fluorescence. However, in the absence of substrates, we did not observe rapid interconversion between the fluorescent and nonfluorescent states comparable to that observed in the presence of substrates; FMN dissociation was generally irreversible. Therefore we attribute FMN reduction as the cause for the short-lived nonfluorescent states when substrates were present. The turnover trajectories of 71 DHODA dimers (408 turnovers in total) were obtained with 50 μM DHO and 10 μM fumarate in 50 mM sodium phosphate buffer, pH 7 at 22°C. Significant heterogeneity in the behavior of individual molecules was observed and they fell into three groups. Twenty-eight of the 71 molecules (40%) interconverted between the three oxidation states (Fig. 2 a); these are denoted group 1. Group 2 consists of molecules that interconverted only between the fully fluorescent and half-fluorescent state (Fig. 2 b), accounting for 40% of the dimers (28 of 71). The remaining 20% of the molecules (15 of 71), denoted group 3, turned over only between fluorescent and nonfluorescent states. Group 3 molecules are not monomers because monomeric DHODA is inactive (8), so that they are either dimers interconverting between the half-fluorescent and nonfluorescent states or dimers with only one FMN bound.

Fig. 2.
Fig. 2.

Fluorescence trajectories of different single DHODA dimers during turnover in 50 μM DHO and 10 μM fumarate in 50 mM sodium phosphate buffer, pH 7 at 22°C. (a) DHODA interconverting between all three fluorescent states (group 1). (b) DHODA interconverting only between the fully fluorescent and half-fluorescent states (group 2). (c) DHODA interconverting between only the fluorescent and nonfluorescent states. The three fluorescent states correspond to the fully oxidized (FMN in both subunit), half-oxidized (FMN in one subunit and FMNH2 in the other subunit), and fully reduced (FMNH2 in both subunits).

Turnover trajectories of another 83 DHODA molecules (551 turnovers in total) were obtained with 50 μM fumarate, a higher oxidant concentration. Heterogeneous turnover behavior was again observed. Forty-eight percent of the molecules interconverted between all three fluorescent states and 42% interconverted only between the fully fluorescent and half-fluorescent states. There were fewer molecules turning over between only the half-fluorescent and nonfluorescent states, accounting for 10% of the observed trajectories, as compared with 21% at 10 μM fumarate.

Kinetics.

The turnover kinetics of single DHODA molecules can be analyzed by plotting the distribution of dwell-times in each of the three fluorescent states, corresponding to the waiting time for reduction or oxidation of FMN in each DHODA subunit. Dwell-time distributions were constructed separately for the three different groups of molecules for both concentrations of fumarate (Fig. 3 and Table 1). The dwell-times in the half-fluorescent state were analyzed in two subsets, depending on whether the molecules were reduced by DHO or oxidized by fumarate.

Fig. 3.
Fig. 3.

Dwell-time distribution in the on-state (oxidized-state) of DHODA molecules with 10 μM fumarate. (a) Fully fluorescent state. (b) Half-fluorescent state (converting to the fully fluorescent state). (c) Half-fluorescent state (converting to the nonfluorescent state). (d) Nonfluorescent state. Group 1 data are denoted by •, group 2 by □, and group 3 by ▵ (see Fig. 2 for definition of the different groups). All distributions are fit to single-exponential decays.

View this table:
Table 1.

Kinetic results for the three groups of DHODA molecules obtained with 50 μM DHO and 10 or 50 μM fumarate

Regardless of which group a molecule was in, the observed rate constant did not vary significantly for any of the four possible state conversions. This was true at both 10 and 50 μM fumarate, although the number of turnovers from the group 3 molecules identified at 50 μM fumarate was too small statistically to fit a dwell-time histogram distribution. Moreover, the observed rate constants for the reduction of the fully oxidized and half-oxidized states were very similar at either 10 or 50 μM fumarate (refer to the two columns listed as reduction in Table 1), as expected for an enzyme with ping-pong kinetics. Increasing the oxidizing substrate concentration from 10 to 50 μM accelerated the rates of oxidation, suggesting that the fumarate concentration was not saturating at 10 μM.

Half-Sites Reactivity.

A simple reaction model with independently reactive subunits is not compatible with the behavior of group 1 molecules described above. The simplest reaction model that accounts for the observed kinetics of DHODA turnover is illustrated in Fig. 4, where reactive subunits are marked with an asterisk and only one subunit is active at a time, e.g., only one subunit of the fully oxidized enzyme (E*oxEox) reacts with DHO. Subsequently the enzyme (E*redEox) is either oxidized by fumarate while the reduced subunit is reactive or it isomerizes to EredE*ox, allowing the oxidized subunit to be reduced. Similarly, the fully reduced enzyme (E*redEred) can be oxidized at only one of the subunits. The resulting half-oxidized enzyme (E*oxEred) is then either reduced at the oxidized subunit or converts to the isomer, EoxE*red, allowing oxidation of the reduced subunit. The key features of the model are that only one subunit of the dimer can react at a time and, when the enzyme is in the half-oxidized state, it isomerizes at a kinetically significant rate between two conformations with either the reduced subunit or the oxidized subunit being active.

Fig. 4.
Fig. 4.

Minimal kinetic scheme for DHODA catalysis. Eox denotes the monomer with FMN in the oxidized state, and Ered denotes FMN in the reduced state. OA, orotate. The reactive subunit is signified with an asterisk. k′1 and k′2 are the net rate constants for reduction and oxidation, respectively, encompassing the binding of substrate at a particular concentration, its reaction in the central complex, and the release of product. γ1 and γ2 are the interconversion rates between the two isomers in the half-oxidized state.

The mechanism can be analyzed to find relationships between the rate constants defined in Fig. 4 and the experimentally observable decay rate constants. Because the fully oxidized state has only one reaction pathway available, the rate constant observed for the decay of the dwell-time distribution of the fully fluorescent state is the net rate constant for reduction, k′1, encompassing DHO binding, reaction, and orotate release. Similarly, the dwell-time distribution of the nonfluorescent state gives the net rate constant for oxidation k′2, encompassing fumarate binding, reaction, and succinate release. The behavior of the half-oxidized state is more complex. There are two forms of half-oxidized enzyme, that having the reduced subunit in the reactive conformation (E*redEox) and that having the oxidized subunit in the reactive conformation (EredE*ox), and they have the same fluorescence. Both half-oxidized forms can react by two competing pathways. One form, E*redEox, is either oxidized by fumarate (k′2) or isomerizes with a rate constant γ1 to allow the reaction of the other subunit with DHO. Similarly, EredE*ox is either reduced by DHO (k′1) or isomerizes (γ2) to the form that can react with fumarate. An analytical solution for the two eigenvalues describing the dwell-time distribution of the half-oxidized states gives two observed rate constants (see Appendix): Embedded Image As the value of γ1 + γ2 becomes small compared with the value of k′1 − k′2, the behavior simplifies to give a decay rate constant approaching the value of k′1 for the reduction of the half-oxidized state and k′2 for its oxidation (see Appendix). A Monte Carlo simulation was used to verify this analysis (data not shown). Hypothetical single-molecule turnover trajectories were generated for the model in Fig. 4 with k′1 set to 20 s−1, k′2 set to 45 s−1, γ1 set to 5 s−1, and values of γ2 ranging from 30 s−1 to 1 s−1. For values of γ2 above ≈10 s−1, a biphasic distribution was obtained for the dwell-times of the half-oxidized molecules that were reduced. For values of γ2 below 10 s−1, dwell-time distributions were single exponentials with observed rate constants of about 20 s−1, showing that when the value of γ2 is sufficiently small, the value of the observed rate constant simplifies as described above.

The single-molecule kinetic results conform to the half-sites reactivity model if the isomerization rates, γ1 and γ2, are sufficiently small. Our data showed similar net rate constants for reduction of all oxidized states (in the range of ≈20–30 s−1; Table 1), suggesting that the rate constant (γ1) for the conversion of E*redEox to EredE*ox is relatively small. The net rate constants for oxidation of the reduced states were also indistinguishable (≈47 s−1 at 10 μM fumarate and ≈63 s−1 at 50 μM fumarate; Table 1), suggesting that the rate constant (γ2) for the conversion of EredE*ox to E*redEox is also small.

The differences in the turnover behavior of the three groups of DHODA molecules can be explained in terms of interconversion rates between the two isomers in the half-oxidized state. For group 1, γ1 and γ2 have similar values and are large enough so that dimers isomerize relatively frequently in the half-oxidized states, causing turnover to occur through both loops in Fig. 4. It is not possible to accurately estimate lower limits for γ1 and γ2 from our data, but our simulation analysis indicates values of γ1 lower than 2 s−1 would cause the formation of the fully reduced enzyme to become too rare to be consistent with the limited length of our single-molecule data. In contrast, γ1 for group 2 dimers is so small that turnover is restricted to E*oxEox and E*redEox (the upper half of the reaction scheme in Fig. 4). Similarly, group 3 dimers turn over only in the lower half of the catalytic cycle because γ2 is much smaller than k′1, k′2, and γ1.

To verify that our conclusions from the simulations of group 1 molecules are consistent with the data, a rough estimate of γ1 was calculated from the ratio of the number of oxidation steps to the number of reduction steps when the enzyme was in the half-oxidized state. In the limit of small γ2, this ratio is on the order of k′2/γ1, and was 3:1, giving a rough value of 15 s−1 for γ1. Similarly, in the limit of small γ1, γ2 was estimated to be 8 s−1 from k′1/γ2 ≈ 2.5:1. It should be emphasized that these values are a crude upper limit because these are not analytical calculations (because of the complexity of the kinetics of coupled reactions), and the number of steps that could be counted in the relatively short trajectories was statistically low. Furthermore, the estimate is an upper bound because only the first part of the time history is used, and this history is biased toward short durations of cycling between the fully oxidized and half-oxidized states. However, this calculation does show the consistency between our simulations and the data.

Other simpler reaction models were considered but failed to account for the kinetic results. For instance, a reaction model was considered in which both subunits are equivalently reactive so that the fully oxidized enzyme can be reduced at either subunit; the remaining oxidized subunit can then be reduced, or the reduced subunit can be oxidized. Molecules becoming completely reduced can be oxidized at either subunit. The dwell-time distributions for this model of the half-fluorescent state would give a decay rate constant of k′1 + k′2 regardless of whether the reaction is a reduction or an oxidation. Moreover, because both subunits are equally active, the dwell-time distribution of the fully fluorescent state would give an observed rate constant of 2k′1, whereas the distribution for the fully nonfluorescent state would give an observed rate constant of 2k′2. The measured rate constants for group 1 molecules do not satisfy these conditions: The net rate constant observed for the reduction of the half-fluorescent state differs from that for its oxidation, the rate constant observed for the reduction of the fully oxidized dimer is not twice that observed for group 2 molecules, and the rate constant for the oxidation of the fully reduced dimer is not twice that observed for group 3 molecules. The simplest model we were able to devise that is consistent with the behavior of the group 1 molecules is the half-sites reactivity model shown in Fig. 4.

Discussion

Our data demonstrate the applicability of single-molecule spectroscopy as a tool to study cooperativity, and they provide insight into the subunit reactivity of DHODA. Isolated dimers show significant heterogeneity in their kinetic behavior during turnover. Less than half of the DHODA molecules turn over between all three oxidation states (fully oxidized, half-oxidized, and fully reduced) under the conditions used, whereas the rest interconvert between only two of the three redox states. Regardless, kinetic analyses gave similar reaction rates for both reduction and oxidation, leading to our half-sites model, which appears to be the minimal model capable of explaining the kinetics. Rapid-reaction studies on DHODA under slightly different conditions (R. L. Fagan, M. N. Nelson, and B.A.P., unpublished data) indicate that the rate constants obtained for the reactions of an ensemble are similar to those obtained by single-molecule spectroscopy, and that fumarate binds with an affinity consistent with the changes caused by varying fumarate concentrations in single-molecule studies. Furthermore, steady-state kinetics using oxidizing substrates other than fumarate (9) gave k cat values consistent with the single-molecule studies. Interestingly, orotate has been suggested to bind in a half-sites fashion (10), and half-sites oxidation by fumarate has been observed (B.A.P., unpublished data).

The structural basis for the observed half-sites reactivity in DHODA is not yet known, but several possibilities can be imagined. One explanation is that accessibility to the active site, controlled by a protein loop, differs between subunits of the dimer, and that movement of the loops is coordinated. Crystal structures of the wild-type enzyme, either without ligands or with orotate bound, did not reveal asymmetry (11, 12). However, ligand-binding studies using orotate showed nonhyperbolic binding and a stoichiometry at saturation of one bound orotate per dimer (10). Also, the crystal structure of the Lys-213 → Glu mutant shows asymmetry between the two subunits (10), with the loop of one subunit in a closed conformation similar to that of the wild-type enzyme, and the other in an open conformation. Catalysis by this mutant was impaired significantly. As an alternative to differing ligand accessibilities, it is possible that the active sites within a dimer differ in the pKa of the active site acid/base. Cys-130, located on the loop that covers the active site, deprotonates DHO during the reductive half-reaction and presumably protonates fumarate in the oxidative half-reaction. These functions require the proper ionization state of Cys-130 when the loop is closed, and it is possible that the ionization state (and hence reactivity) of the cysteine of one subunit depends on the state of the other subunit.

In summary, our single-molecule results for DHODA, a homodimer, demonstrate the potential of using the single-molecule approach to derive detailed kinetic information on subunit activity and interaction. We have shown that single-molecule studies can reveal not only static heterogeneity in the catalytic activities of oligomeric proteins, but more importantly, provide a means for investigating catalytic cooperativity. This analysis could be applied to study other complicated flavoenzymes, such as heterodimers or higher oligomers. Single-molecule spectroscopy is a promising approach in providing complementary kinetic and mechanistic information that is beyond the scope of conventional ensemble enzymology.

Materials and Methods

Materials.

Wild-type DHODA from L. lactis was expressed in Escherichia coli strain SØ6645 and purified by ion-exchange chromatography, according to the procedures of Nielsen et al. (13), giving enzyme of high purity and specific activity similar to ref. 13. DHO and fumaric acid were purchased from Sigma.

Confocal Fluorescence Microscopy.

The setup of our inverted confocal microscope has been described in ref. 14. In brief, the 457-nm line of an Ar+-ion laser is expanded and reflected by a dichroic mirror. The laser beam is then focused by a high numerical aperture (NA) oil-immersion objective (NA = 1.65, Olympus) on a coverslip coated with the sample under study. The sample fluorescence is collected by the same high-NA objective and detected by a single-photon-counting avalanche photodiode. The sample slide is mounted on a piezo scanning stage, which precisely moves the stage for scanning different locations of the sample. The image of the sample is reconstructed by raster scanning. When the fluorescence of the local area was higher than a threshold set at 8–10 counts per bin, trajectories were collected with time points at intervals of 10 ms.

Sample Preparation for Single-Molecule Studies.

Single DHODA molecules were immobilized in the pores formed in a 1% agarose gel as described (14, 15). Low gelling-point agarose (Sigma, type 4) was melted in 50 mM sodium phosphate buffer, pH 7. DHODA molecules were diluted into the agarose solution to 1 nM just above the gelling temperature (≈30°C), and the mixture was spun on a coverslip at 2,000 rpm for 10 s in a Photo-resist Spinner model 1-EC101-CB15 (Headway Research, Garland, TX), forming a smooth thin layer of gel containing the enzyme molecules. After the coverslip was mounted on the scanning stage of the microscope, a small volume of buffer (≈120 μl) was applied on the sample. In turnover experiments, the substrates were premixed with the agarose solution. It was noted that subunit dissociation could be significant with sample concentrations in the nanomolar range. Nonetheless, single DHODA dimers can be distinguished from monomers because dimers with two bound flavins exhibited fluorescence drops in two distinct drops in intensity to the background level, reflecting the loss of fluorescence as each FMN dissociates (Fig. 1). The fluorescence of monomers dropped in one step to the background level, because of the fluorescence loss of the single FMN.

Data Analysis.

Dwell-time histograms were constructed from single-molecule trajectories of multiple molecules because individual reaction trajectories were too short and did not contain enough turnovers for statistical analysis. The three distinct fluorescence states were differentiated by considering the distinct levels of fluorescence intensity, whose difference is significantly higher than shot noise. The interconversions between the different fluorescent states were determined by evaluating the variance of the fluorescence fluctuation. If fluorescence fluctuations (quantified by standard deviation of the fluorescence intensity) were significantly larger than the Poisson noise, we regarded them as conversion steps between fluorescence states.

Appendix

The dwell-time distribution of the half-fluorescent molecules going into the fully fluorescent state is f ro = k′2 P ro, where P ro(t) is the conditional probability that the molecule is in state E*redEox at time t, given it is in the half-oxidized state between 0 and t. The dwell-time distribution of the half-fluorescent molecules going into the nonfluorescent state is f or = k′1 P or, where P or(t) is the conditional probability of being in state EredE*ox. P ro and P or are determined by the following coupled differential equations, Embedded Image and Embedded Image The eigenvalues of the above equations are Embedded Image If the molecule is initially in E*redEox at time 0, P ro(0) = 1 and P or(0) = 0. With such initial conditions, P ro(t) is solved as Embedded Image If the molecule is initially in EredE*ox at time 0, P ro(0) = 0 and P or(0) = 1. With such initial condition, P ro(t) is solved as Embedded Image Hence, according to ref. 16, P ro(t) = N −1[k′1ρoo·P ro,1(t) + k′2ρrr·P ro,2(t)], where ρoo and ρrr are the time-independent equilibrium distribution of the molecule being in state E*oxEox and E*redEred, respectively, and N = (k′1ρoo + k′2ρrr) is the normalization factor. k′1ρoo is the equilibrium flux from state E*oxEox to E*redEox, and k′2ρrr is the equilibrium flux from state E*redEred to EredE*ox. The equilibrium distribution is Embedded Image where C = (k′1 + k′2)(k′1γ′1 + k′2γ′2).

When γ1 + γ2 is much smaller than the value of k′1 − k′2, λ1 ≈ −k′1, λ2 ≈ −k′2 and P ro(t) ≈ N −1 k′1ρoo e −k′2t. A similar rationale can be applied to derived P or(t). In the limit of small γ1 and γ2, P or(t) ≈ N −1 k′1ρoo e −k′1t.

Acknowledgments

This work was supported by National Institutes of Health Grants GM61087 (to B.A.P.) and AG018017 (to A.G. and D.S.) and by Michigan Life Science Corridor (MLSC) Proposal 1537 (to A.G. and D.S.).

Footnotes

  • ‖To whom correspondence should be addressed. E-mail: brupalf{at}umich.edu

  • Author contributions: J.S., A.G., D.S., and B.A.P. designed research; J.S. performed research; J.S. and J.D. contributed new reagents/analytic tools; J.S., A.G., D.S., and B.A.P. analyzed data; and J.S., A.G., D.S., and B.A.P. wrote the paper.

  • Conflict of interest statement: No conflicts declared.

  • This paper was submitted directly (Track II) to the PNAS office.

  • Abbreviations:

    Abbreviations:

    DHO,
    dihydroorotate;
    DHODA,
    DHO dehydrogenase A.
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Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis
Jue Shi, Joe Dertouzos, Ari Gafni, Duncan Steel, Bruce A. Palfey
Proceedings of the National Academy of Sciences Apr 2006, 103 (15) 5775-5780; DOI: 10.1073/pnas.0510482103
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