Supplementary Information

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1 Supplementary Information Adenosyltransferase Tailors and Delivers Coenzyme B 12 Dominique Padovani 1,2, Tetyana Labunska 2, Bruce A. Palfey 1, David P. Ballou 1 and Ruma Banerjee 1,2 * 1 Biological Chemistry Department, University of Michigan, Ann Arbor, MI and 2 Redox Biology Center and Department of Biochemistry, University of Nebraska, Lincoln, NE

2 Figure S1. Binding of AdoCbl to MCM. Representative kinetic traces (black) for the binding of AdoCbl (11, 23 and 40 µm, bottom to top respectively) to apo-mcm (7 µm after mixing) in 50 mm KPi buffer, ph 7.5 at 20ºC. The kinetic simulations (in red) were generated using the model shown in Fig. S5a (upper) and the parameters k 1 -k 6 for the dissociative mechanism shown in Table SI. Inset: Dependence of the k obs for AdoCbl binding to MCM ( ) on cofactor concentration. The red line represents the simulated dependence of k obs on AdoCbl and yields values for the slope and intercept of µm -1 s -1 and 0.49 s -1 respectively, similar to the experimentally determined values of 0.10±0.02 µm -1 s -1 and 0.26±0.05 s -1. Based on the observed ΔA 525nm (Δε of 1.06 mm -1 cm -1 ), one AdoCbl is bound per heterodimer of MCM.

3 Figure S2. Properties of ATR from M. extorquens AM1. (a) The oligomeric state of ATR in solution was determined by size exclusion chromatography performed as described under Supplementary Methods. The molecular weights of the standards (solid line) are: thyroglobulin (670 kd), γ-globulin (158 kd), ovalbumin (44 kd), myoglobin (17 kda) and vitamin B 12 (1.35 kd). The retention time of 52.0 ± 0.2 min for ATR (dashed line) yields a native molecular mass of 55.2 ± 1.1 kd, corresponding to a homotrimer. (b) Scatchard analysis of AdoCbl binding to apo-atr reveals the presence of two nonequivalent AdoCbl binding sites per ATR trimer, with n 1 = 1.3 ± 0.3, K D1 = 0.12 ± 0.03 µm and n 2 = 1.99 ± 0.10,

4 K D2 = 1.0 ± 0.16 µm (n=3). Inset: Titration of AdoCbl (34.7 µm) with increasing concentrations of apo-atr ( µm) as described under Supplementary Methods. Representative traces are shown for clarity. (c) ITC titration of apo-atr (30.6 µm) in 50 mm KPi buffer, ph 7.5, 300 mm KCl with aliquots of a 1.0 mm stock solution of AdoCbl. The top panel shows the raw data in power versus time. The area under each spike is proportional to the heat produced with each injection. The lower panel shows the integrated areas normalized to the number of moles of AdoCbl added with each injection. Data were well-fitted to a two-site binding model and yielded values of K D1 = 0.6 ± 0.1 µm and K D2 = 1.5 ± 0.4 µm respectively. (d) Representative kinetic traces (in black) for the binding of AdoCbl (10-30 µm top to bottom) to apo-atr (5 µm). The simulations (in red) were performed using the model described in Fig. S5a (lower) and using the kinetic parameters (k 7 -k 14 ) for the dissociative model shown in Table SI. Inset: Dependence of k obs1 ( ) and k obs2 ( ) for binding of AdoCbl on cofactor concentration. A linear fit to the experimental data yields the following values: slope 1 =3.4±0.4 µm -1 s -1, intercept 1 =44±9 s -1, slope 2 =1.3±0.4 µm -1 s -1 and intercept 2 =13±3 s -1. The simulated data (in red) yield comparable values: slope 1 ~3.75 µm -1 s -1, intercept 1 ~30.2 s -1, slope 2 ~1.18 µm -1 s -1 and intercept 2 ~24.9 s -1. The data are represented as mean ± S.D.

5 Figure S3. Kinetics of interprotein AdoCbl transfer. (a) A representative set of stopped-flow scans for the transfer of AdoCbl between holo-atr (9.5 µm AdoCbl after mixing) and wildtype apo-mcm (90 µm, after mixing) in 50 mm KPi buffer, ph 7.5, at 20ºC. Traces were recorded every 0.5 sec. The arrows denote the direction of absorption changes. (b) Representative stopped-flow traces observed for AdoCbl transfer between holo-atr (9.6 µm bound AdoCbl, after mixing) and wild-type apo-mcm (67.5 µm, after mixing) (trace 1) or between holo-mcm (15 µm, after mixing) and apo-atr (38.5 µm, after mixing) (trace 2). The red lines represent biphasic fits (R 2 ~0.998) to the experimental data. In the range of concentrations studied, transfer of AdoCbl occurs with changes in amplitude ΔA 1 ~0.9-1ΔA 2 and ΔA 1 ~ ΔA 2, in the forward and reverse directions, respectively. The k obs1 and k obs2 values obtained from the kinetic traces at each concentration of apo-[e2] were then used to generate Figs. S4a and b.

6 Figure S4. Dependence of AdoCbl transfer kinetics on the concentration of the acceptor protein. (a) Hyperbolic dependence of k obs1 and k obs2 (inset) for AdoCbl transfer from holo- ATR on the concentration of apo-mcm. The values for forward transfer k trans+1 and k trans+2, were obtained as described under Supplementary Methods. We note that the k obs values are not true rate constants but eigenvalues that are composites of individual rate constants. The eigenvalues appear to decrease because the amplitudes become larger at increasing concentrations of E2 and it therefore takes longer to make the complete transfers. (b) Hyperbolic dependence of k obs1 and k obs2 (inset) for AdoCbl transfer from holo-mcm on the concentration of apo-atr. The values for reverse transfer (k trans-1, k trans-2 ) were obtained from these plots as described under Supplementary Methods. The data are represented as mean ± S.D.

7 Figure S5. Alternative mechanisms for AdoCbl transfer. Equations describing the dissociative (a) and associative (b) models for cofactor binding to and transfer between ATR and MCM.

8 Figure S6. Temperature dependence of AdoCbl binding to MCM. Comparison of the temperature dependence of the observed rates of transfer k trans+1 ( ) and k trans+2 ( ) from holo-atr (10 µm) to apo-mcm (100 µm) and of the binding of AdoCbl (50 µm) from solution to apo-mcm (10 µm) ( ) in 50 mm KPi buffer, ph 7.5. The data are represented as mean ± S.D.

9 Table S1 Kinetic parameters obtained from simulations of the dissociative and associative mechanisms. Dissociative mechanism Associative mechanism Kinetic parameters K D Kinetic parameters K D k 1, µm -1 s } 43.5 µm k 1, µm -1 s k 2, s k 2, s } 7.05 µm k 3, s k 3, s k 4, s k 4, s k 5, s k 5, s k 6, s k 6, µm -1 s k 7, µm -1 s k 7, µm -1 s } 69.4 µm k 8, s k 8, s } 4.35 µm } 0.36 µm k 9, s k 9, s k 10, s k 10, s k 11, µm -1 s k 11, s } 0.37 µm k 12, s k 12, µm 1 s } 2.19 µm k 13, s k 14, s -1 20

10 Table S2 Comparison of the activation parameters for transfer of AdoCbl from holo-atr to apo-mcm and for the binding of AdoCbl to apo-mcm from solution. Parameters Site 1 a Site 2 a solution E a, kcal/mol 10.3± ± ±0.9 ΔH, kcal/mol 9.7± ± ±0.9 ΔS, cal/(mol.k) -24.4± ± ±3.2 ΔG, kcal/mol 16.8± ± ±0.1 a The sites refer to the two binding sites for AdoCbl on ATR.

11 Table S3 Kinetic and thermodynamic parameters for the H596N/A mutants of MCM. a Parameters Wild-type b H596A H596N k cat, s ± ± ±0.006 K M-[R]MCoA, µm 86±13 107±32 64±21 k cat /K M, M -1 s -1 (1.53±0.19) ±6 322±75 K act-adocbl, µm 1.6±0.1 c 4.5± ±0.3 K D, µm d 0.40 ± ± ± 1.7 a The kinetic parameters for MCM were determined using the radiolabeled assay at 37 C as described under Methods and represent the average (± S.D.) of three independent experiments. b From Padovani, D., and Banerjee, R. (2006) Biochemistry 45, except for (c), which was determined as described under Methods. d Determined by ITC (in 50 mm KPi, ph 7.5, containing 300 mm KCl, at 20 C) as described under Supplementary Methods.

12 Supplementary Methods Materials. AdoCbl and other reagent grade chemicals were purchased from Sigma. [ 14 C]- CH 3 -malonyl-coa (56 Ci/mol) was purchased from New England Nuclear. Construction of Site-Specific Mutants. The plasmid containing the M. extorquens AM1 MCM and ATR were a generous gift from Mary E. Lidstrom (University of Washington, Seattle). The site-directed mutants H596N and H596A were created using the QuickChange kit (Stratagene) and the following sense primers: 5 - CAAGATGGGCCAGGACGGGAACGACCGCGGCCAGAAGGTG-3 for H596N and 5 - CAAGATGGGCCAGGACGGGGCCGACCGCGGCCAGAAGGTG-3 for H596A. The antisense mutagenic primers had the complementary sequences. Following PCR amplification, the mutations were confirmed by nucleotide sequence determination at the Genomics Core Facility (University of Nebraska-Lincoln). Enzymes Expression and Purification. The wild-type and mutant MCMs 1 and ATR 2 were purified as previously described. Enzyme assays. The specific activity of MCM was determined in the radiolabeled assay as described previously. 3 Each experiment was performed in triplicate. (i) Kinetic parameters for wild-type and mutant MCM. The kinetic parameters for the mutants, H596A/N, were determined at 37 C by increasing the duration of the assay from 3 to 10 min and the amount of enzyme 200- to 1,000-fold. K M and V max were determined in the presence of varying concentrations of (R,S)-[ 14 C]-methylmalonyl-CoA ( µm) while keeping constant the concentration of AdoCbl at 100 µm in the assay. The K act for AdoCbl was obtained by varying the concentration of the cofactor (1-50 µm) in the presence of a saturating concentration of the substrate (4 mm (R,S)-[ 14 C]-methylmalonyl- CoA).

13 (ii) Modulation of MCM activity by ATR. To assess MCM activity under conditions where the percent cofactor transfer was monitored spectroscopically, the concentration of enzymes in the assay had to be increased ~1,000-fold. As a consequence, after the AdoCbl transfer experiments, the activity measurements were performed on ice to be under initial velocity conditions in the radiolabeled MCM assay. AdoCbl transfer was accomplished at 20ºC for 10 min by incubating in the forward direction, 5 µm holo-atr (10 µm bound AdoCbl) with various concentrations of apo-mcm ( µm) and in the reverse direction, 10 µm holo-mcm (containing a stoichiometric amount of AdoCbl) with varying concentrations of apo-atr (0-50 µm final concentration), in 50 mm potassium phosphate (KPi) buffer, ph 7.5. Then, the solutions were incubated for 20 min on ice before starting the assay by addition of 5 mm ice-cold (R,S)-[ 14 C]-methylmalonyl-CoA. After 1 min incubation, the reaction mixtures were quenched and the samples treated as previously described. 3 Oligomeric State of ATR. To determine the oligomeric state of apo-atr in solution, ~0.2-1 mg of the enzyme was loaded on a 2 x 70 cm Sephacryl 200 column in 50 mm KPi, ph 7.5, containing 100 mm KCl at a flow rate of 2 ml min -1. Prior to loading, the protein was filtered through a 0.2-µm Anotop 10 filter (Whatman). The column was calibrated using gel filtration standards from Bio-Rad. AdoCbl binding to ATR: UV visible spectroscopy. The large blue shift in the UV-visible spectrum (from 525 to 458 nm) accompanying binding of the free cofactor to ATR was monitored to determine the binding affinity of ATR for AdoCbl. Briefly, successive aliquots of apo-atr (1-45 µm) were added to a fixed concentration of AdoCbl (20-35 µm). Following each addition of apo-atr, the solution was incubated at 20 C for 10 min prior to recording of the UV-visible spectrum. The concentrations of bound (L b ) and unbound (L u ) AdoCbl after each addition of apo-atr were determined using equations 2 and 3.

14 L b =(ΔA 525nm /ΔA 525nm max)*[adocbl] total [2] L u =[AdoCbl] total -L b [3] The values from equations 2 and 3 were used to obtain the parameters, K D and n (the number of binding sites) using the Scatchard plot described by equation 4. The experiment was performed in triplicate. ([L b ]/[E]) = ( n " [L b ]/[E]) [L u ] 1 K D [4] Since the Scatchard plot was clearly biphasic, each phase was analyzed independently (Fig. S2b). Isothermal titration calorimetry (ITC). All calorimetric binding experiments were performed as described previously. 1 Each experiment was performed at least in triplicate and the data were analyzed using Microcal ORIGIN software. (i) Binding of AdoCbl to apo-atr. Apo-ATR (10-45 µm in different experiments) was titrated with fifty nine 5 µl aliquots of a µm solution of AdoCbl in 50 mm KPi buffer, ph 7.5 at 20.0 ± 0.1 C. When the same experiments were performed with buffer containing 300 mm KCl, no significant difference was observed. The calorimetric signals were integrated, and the data fit well to a two-site binding model to estimate the equilibrium association constants, K A, and the binding enthalpies, ΔH. The Gibbs free energy of binding, ΔG, and the entropic contribution to the binding free energy, -TΔS, were calculated using equations 5 and 6. ΔG = -RT ln K A [5] ΔG = ΔH - TΔS [6]

15 (ii) Binding of AdoCbl to MCM mutants. Enzyme (10-50 µm of H596N/A mutants of MCM) in 50 mm KPi buffer, ph 7.5, was titrated with twenty nine 10 µl aliquots of a µm solution of AdoCbl at 20.0 ± 0.1 C. When the same experiments were carried out in buffer containing 300 mm KCl, very small changes ( 6%) in the binding parameters were observed. The data were analyzed as described for ATR but using a single-site binding model. AdoCbl transfer between ATR and MCM. The transfer of AdoCbl between the two enzyme active sites (in 50 mm KPi buffer, ph 7.5) was monitored by absorption spectroscopy at 20ºC. In the forward direction, 7 µm holo-atr (14 µm bound AdoCbl) was added to a final concentration of µm apo-mcm (wild-type or mutant). In the reverse direction, apo- ATR (3-60 µm final concentration) was added to 15 µm wild-type holo-mcm (containing 15 µm bound AdoCbl). The amount of AdoCbl transferred was estimated from the increase (forward) or decrease (reverse) in absorbance at 525 nm (Δε 525 nm = 7.75 mm -1 cm -1 for wild-type MCM and Δε 525 nm = 5.46 mm -1 cm -1 for the mutants). Stopped-flow spectroscopy. Stopped-flow UV-visible spectroscopy experiments were performed on an Applied Photophysics spectrophotometer (ISX.MV18) under red light illumination. Rapid-scanning stopped-flow fluorescence kinetic measurements were made using an OLIS stopped-flow fluorescence spectrophotometer. Fluorescence emission spectra were recorded between 325 and 474 nm using an emission slit width of 0.6 µm and acquired at 62 scans/s. The excitation wavelength was 282 nm and two consecutive slits of 0.6 µm width were used, as reported previously. 4 All solutions were prepared in 50 mm KPi buffer, ph 7.5, filtered through a 0.02 µm syringe filter (Whatman), transferred to the loading syringes and allowed to equilibrate for at least 20 min before initiating the experiments. An external water bath was used to maintain the loading syringes and the mixing chamber at 20 ± 0.5 ºC.

16 (i) Kinetic analysis of AdoCbl binding to apo-mcm. The kinetic parameters for binding of AdoCbl to apo-mcm were determined by mixing AdoCbl ( µm before mixing) and a fixed concentration of apo-mcm (10-15 µm before mixing in different experiments) in 50 mm KPi buffer, ph 7.5, and monitoring the increase in A 525nm (Δε 525 nm = 1.06 mm -1 cm -1 ). Kinetic traces were well-fitted to a single-exponential function to obtain k obs and the change in amplitude, ΔA, according to equation 7, where A is the absorbance at time t and A 0 is the offset for the exponential increase. A = A o + "A(1# e (#k obs t ) ) [7] The k obs value was then plotted as a function of AdoCbl concentration. The kinetic parameters for binding of AdoCbl to apo-mcm were also determined by mixing AdoCbl (2-15 µm after mixing) and a fixed concentration of apo-mcm (0.5-1 µm after mixing in different experiments) in 50 mm KPi buffer, ph 7.5, and monitoring the quenching of fluorescence at 340 nm. Kinetic traces were well-fitted to a single-exponential function to obtain k obs and the change in fluorescence, ΔF, according to equation 8, where F is the absorbance at time t and F 0 is the offset for the exponential decrease and c is the rate of background fluorescence decay. F ( = F + " F(1! e o! kobs t) )! ct [8] The rate of background fluorescence decay (c) was recorded by mixing µm apo- MCM (after mixing) against buffer. The k obs value was then plotted as a function of AdoCbl concentration. (ii) Kinetic analysis of AdoCbl binding to apo-atr. Binding of AdoCbl to apo-atr was carried out as described above for MCM by rapidly mixing varying concentrations of AdoCbl ( µm before mixing) with a fixed concentration of apo-atr (5-16 µm before

17 mixing) in 50 mm KPi buffer, ph 7.5, and monitoring the decrease in A 525nm (Δε 525 nm = -6.7 mm -1 cm -1 ). Kinetic traces were best fitted to a double exponential function to obtain k obs1 and k obs2 and the changes in amplitude associated with each phase, ΔA 1 and ΔA 2, as described in equation 9, where A is the absorbance at time t and A 0 is the offset for the exponential decrease. A = A o + "A 1 e (#k obs1t ) + "A 2 e (#k obs 2t ) [9] Then, the k obs (k obs1 and k obs2 ) values were each plotted as a function of AdoCbl concentration and the plots were fitted to a linear function to obtain the slope and intercept for each binding site. (iii) AdoCbl transfer experiments. Cofactor transfer in the forward direction (from holo-atr to apo-mcm) was investigated by rapidly mixing a fixed concentration of holo-atr (10-20 µm before mixing) with increasing concentrations of apo-mcm (3-180 µm before mixing). The concentration of AdoCbl bound to ATR was determined spectrophotometrically (ε 458 nm = 8 mm -1 cm -1 ). Transfer was monitored at 525 nm (Δε 525nm ~ 7.75 mm -1 cm -1 ) or 458 nm (Δε 428nm ~ mm -1 cm -1 ). Kinetic traces were best fitted to a double exponential function using equation 9, when monitoring the transfer at 458 nm, or equation 10, when monitoring the transfer at 525 nm, to obtain both the observed rates of transfer (k obs1 and k obs2 ) and the change in amplitude (ΔA 1 and ΔA 2 ) associated with each phase. A = A o + "A 1 (1# e (#k obs1t ) ) + "A 2 (1# e (#k obs 2t ) ) [10] In this equation, A represents the absorbance at time t and A 0 the offset for the exponential increase. The dependence on apo-mcm concentration associated with the transfer of AdoCbl from each binding site on holo-atr was then obtained by plotting the observed rates of transfer (k obs1 and k obs2 ) as a function of apo-mcm concentration and by fitting the

18 data to a hyperbolic decay function, yielding the values for k trans+1 and k trans+2 at saturating concentrations of apo-mcm. At least 6 independent traces were recorded at each apo-mcm concentration and the experiment was performed in duplicate. The reactions were also monitored in a scanning mode with a photodiode array detector. A total of scans were collected over a range of sec using the program XScan (Applied Photophysics) and analyzed using Sigma Plot. We note that in the scanning mode, photolysis of cofactor occurs after ~6 sec. To monitor the transfer of AdoCbl in the reverse direction, varying concentrations of apo- ATR (4-160 µm before mixing) and a fixed concentration of holo-mcm (20-30 µm before mixing, based on the bound AdoCbl concentration using ε 525 nm = 9.06 mm -1 cm -1 ) were rapidly mixed and absorbance changes were monitored at 458 nm or 525 nm. The kinetic traces were well-fitted to a double exponential function using equations 9 and 10. The k trans- 1 and k trans-2 values for the reverse transfer of AdoCbl from holo-mcm to each binding site of apo-atr were estimated at saturating concentrations of apo-atr, as described above. (iv) Thermodynamic parameters for AdoCbl transfer- The activation parameters for the transfer of AdoCbl from holo-atr were obtained from the temperature dependence of the k trans+1 and k trans+2 between 4-28ºC. Holo-ATR (20 µm before mixing) and apo-mcm (200 µm before mixing) were rapidly mixed and absorption change at 525 nm was monitored. The enthalpy (ΔH ) and the entropy of activation (ΔS ) were obtained from the slope and intercept, respectively, of the Eyring plot (equation 11), where where k B is the Boltzmann ln(k transfer /T) = ln (k B /ħ) - ΔH /RT + ΔS /R [11] constant, ħ is Planck's constant and R is the molar gas constant. The Gibbs energy of activation was then obtained at a given temperature, T, using equation 12. ΔG = ΔH - TΔS [12]

19 The activation parameters for binding of AdoCbl (100 µm before mixing) to apo-mcm (20 µm before mixing) were determined between 7 and 32 C as analyzed as described above. Kinetic Simulations- The kinetic simulation program, Berkeley Madonna ( was used to fit the cofactor binding data for the individual enzymes and to distinguish between the dissociative versus associative mechanisms for cofactor transfer between ATR and MCM (described in Fig. S5). All curve fittings were performed using the Runge Kutta 4 method. To simulate the data for cofactor binding to the individual enzymes, 3-5 representative experimental traces were employed (i) Cofactor Binding to MCM. Cofactor binding to MCM was simulated using the equations described in Fig. 5a (k 1 -k 6 ), which describes a model for B 12 binding that has been reported previously ( Model B described in Chowdhury, S., and Banerjee, R. Biochemistry 38, (1999)). The salient steps in the model include: formation of a pre-docking complex of enzyme and cofactor (MCM B 12 )*, followed by a ph-sensitive step (with a pk a ~7.3) to give the protonated complex, (MCM B 12 H + )*. A subsequent conformational change results in docking of the cofactor and enzyme (MCM B 12 H + ). For the simulations, the following assumptions were made and the kinetic parameters were allowed to float: (i) the macroscopic K D (k 2 /k 1 ), for the pre-docking complex, is high ( µm range); (ii) the ph sensitive step is slow (k 4 >k 3 ) and (iii) cofactor docking after the phsensitive step is fast (k 5 >>k 6 ). The simulated traces were fitted to equation 7 and the dependence of the rate constant for AdoCbl binding on cofactor concentration was compared to the experimental data (Fig. S1, inset). An excellent correspondence was observed between the simulated and experimental data (Fig. S1), supporting the validity of the model for B 12 binding to MCM shown in Fig. S5a.

20 (ii) Cofactor Binding to ATR. Cofactor binding to ATR was simulated using equations k 7 -k 14 in Fig. S5a. The salient feature of the model is that it describes negative cooperativity between the two binding sites for B 12 in ATR. A conformational change is shown to follow the binding of each equivalent of B 12. The simulations were subjected to the following assumptions and the kinetic parameters were allowed to float: (i) the first conformational change is very fast (k 9 high); (ii) the first conformational change affects the second conformational change that will then become the rate-limiting step for the binding of the second ligand (k 13 <k 14 ); (iii) the macroscopic K D (k 8 /k 7 and k 12 /k 11 ), for the pre-docking complexes are in the µm range. The simulated kinetic traces were fitted to equation 9 above and the k obs (k obs1 and k obs2 ) values were plotted as a function of AdoCbl concentration to determine the slope and intercept values for the binding of the first and second equivalent of B 12 (Fig. S2d, inset). The correspondence between the experimental and simulated kinetic parameters (Fig. S2d) provides support for the validity of the model describing cofactor binding to ATR. (iii) Dissociative Mechanism. The excellent correspondence between the experimental and simulated data for cofactor binding to the individual enzymes, ATR and MCM described above, provided confidence in the simulated kinetic parameters that were then employed to predict the behavior of the system when the cofactor was transferred from one enzyme to the other via a dissociative mechanism. The values for the first and second order rate constants for binding of B 12 to MCM and ATR (k 1 -k 14 ) described in Table SI were used to fit the dissociative model described in Fig. S5a. Five kinetic traces (three in the forward direction and two in the reverse direction) were fitted to the model (Fig. 2a). (iv) Associative Mechanism. For the associative mechanism, the same five experimental traces used for the dissociative mechanism, were fitted to the model described by equations k 1 -k 12 in Fig. S5b. The salient features of the associative model

21 are: (i) unlike the dissociative model, a rate-limiting ph-sensitive step is not involved, (ii) cofactor transfer occurs in a step-wise manner and thus, a protein-protein complex forms between one ATR and one MCM at a time, (iii) the K D values for complex formation are high, in the micromolar range, consistent with the complex not being detectable by gel filtration chromatography or in a native gel; (iv) the on-rates are higher in the reverse direction than in the forward direction (k 1, k 7 < k 6, k 12 ), since under our experimental conditions, the reverse transfer (from holo-mcm to apo-atr) is favored; (v) the initial estimates for the kinetic parameters were: k 5 =k 11 =100 s -1 and k 6 =k 12 =10 µm -1 s -1, k 2 =k 8 =10 s -1 and k 1 =k 7 =2 µm -1 s -1 ; (vi) the rates of cofactor transfer are faster in the reverse than in the forward direction (k 10 >k 9 and k 4 >k 3 ). The parameters obtained from the initial simulation run were then adjusted manually using the sliders in the software package to obtain values that yielded better overall fits to the experimental data (Fig. 2b) and are reported in Table SI. References for supporting methods 1. Padovani, D., Labunska, T. & Banerjee, R. J. Biol. Chem. 281, (2006). 2. Yamanishi, M., Labunska, T. & Banerjee, R. J Am Chem Soc 127, (2005). 3. Taoka, S., Padmakumar, R., Lai, M.-t., Liu, H.-w. & Banerjee, R. J. Biol. Chem. 269, (1994). 4. Chowdhury, S. & Banerjee, R. Biochemistry 38, (1999).