Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate

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Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism Lauren E Westerhold, Stephanie L Adams, Hanna L Bergman, and Tonya Nicole Zeczycki Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00372 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 7, 2016

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Biochemistry

Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism Lauren E. Westerhold†‡, Stephanie L. Adams†‡, Hanna L. Bergman†‡ and Tonya N. Zeczycki†‡*

†

Department of Biochemistry and Molecular Biology and the ‡East Carolina Diabetes and

Obesity Institute, Brody School of Medicine at East Carolina University, Greenville, NC, 27834

ACS Paragon Plus Environment

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ABSTRACT. Protein structure, ligand binding and catalytic turnover contributes to the governance of catalytic events occurring at spatially distinct domains in multifunctional enzymes. Coordination of these catalytic events partially rests on the ability of spatially discrete active sites to communicate with other allosteric and active sites on the same polypeptide chain (intramolecular) or on different polypeptide chains (intermolecular) within the holoenzyme. Often, communication results in long-range effects on substrate binding or product release. For example, pyruvate binding to the carboxyl transferase (CT) domain of pyruvate carboxylase (PC) increases the rate of product release in the biotin carboxylase (BC) domain. In order to address how CT domain ligand occupancy is “sensed” by other domains, we generated functional, mixed hybrid tetramers using the E218A (inactive BC domain) and T882S (low pyruvate binding, low activity) mutant forms of PC. The apparent Ka pyruvate for the pyruvate-stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that for the T882S homotetramer. In addition, the ratio of the rates of oxaloacetate formation to Pi release for the WT:T882S[1:1] and E218A:T882S[1:1] hybrid tetramercatalyzed reactions was 0.5 and 0.6, respectively, while the T882S homotetramer exhibited a near 1:1 coupling of the two domains, suggesting that the mechanisms coordinating catalytic events is more complicated that we initially assumed. The results presented here are consistent with an intermolecular communication mechanism, where pyruvate binding to the CT domain is “sensed” by domains on a different polypeptide chain within the tetramer.


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Positioned at the crossroads of central metabolism, regulated pyruvate carboxylase (PC, E.C. 6.4.1.1.) activity is crucial to anaplerosis, gluconeogenesis and intermediary metabolite synthesis 1, 2

. Aberrant PC activity contributes to the persistent fasting hyperglycemia characteristic of type

2 diabetes2, 3. Regardless of the metabolic pathway or tissue type, PC catalyzes the MgATPdependent carboxylation of pyruvate by HCO3- to form oxaloacetate in two distinct steps at two spatially discrete active sites (Fig 1)1, 2, 4. In α4 PCs, the four functional domains are contained on a single, ~125-130 kDa MW polypeptide chain, where the overall tetrameric arrangement of monomers creates two distinct catalytic faces4, 5. Two monomers are arranged antiparallel to each other on a single face and perpendicularly to the two additional monomers on the opposing face (Fig 1B). During catalytic turnover, a biotin cofactor, covalently tethered to the biotin carboxyl carrier protein (BCCP) domain, is initially carboxylated in the biotin carboxylase (BC) domain by HCO3- to form a carboxybiotin intermediate. Through the intermolecular translocation of the BCCP domain, the intermediate is shuttled to a neighboring carboxyl transferase (CT) domain active site on an opposing polypeptide chain ~65 Å away (Fig 1C)6-9. Pyruvate is subsequently carboxylated by the CO2 released upon carboxybiotin decarboxylation in the CT domain to generate oxaloacetate. In many cases, the presence of acetyl-CoA in the allosteric domain5, also known as the PC tetramerization domain 9, 10, is required for full activity. Even though the kinetic mechanisms for the BC and CT domain reactions are welldefined6, 11-15, the mechanism underlying the coordination of catalysis between the active sites and the associated global conformational changes associated with efficient turnover remains obscured. Kinetic data16-18 show that CT domain ligand occupancy, by either substrate or substrate analogs, markedly accelerates the release of Pi and MgADP from the BC domain, suggesting established communication between the spatially discrete BC and CT domains within


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the tetramer. In the presence of pyruvate, the increased rate of release of Pi from the BC domain is purported to arise from the substrate-induced BCCP translocation away from the B-subdomain lid of the BC domain to the CT domain on an opposing polypeptide chain6, thereby accelerating the rate of Pi release by facilitating the dynamic translocation of the BCCP domain7, 13, 19, 20 and resulting in efficient CO2 transfer between the two active sites (Fig 1 C). In fact, previous kinetic studies have suggested that a significant amount of Pi is released only upon pyruvate binding to the CT domain19, indicating that conformational changes or linked thermodynamic binding events associated with the presence of pyruvate in the CT domain is the driving force behind the translocation of the BCCP domain and, ultimately, the coordination of catalytic events occurring in the BC and CT domains. Recent Cryo-EM data obtained for PC tetramers actively undergoing catalytic turnover intimates that the enzyme undergoes catalytically relevant global asymmetric-to-symmetric transitions that correlate with the relative positioning of the BCCP domain in the BC or CT domain, respectively9. This data has further shed light into the possibility that PC may be operating under a half-of-the-sites reactive type mechanism, where only a single face of the tetramer is active at any one time. Typical of half-of-the-sites mechanisms, conformational changes associated with product release from the CT or BC domains on one face of the tetramer could possibly promote substrate binding in the other face, resulting in a considerable enhancement of activity when compared to the individual subunits21. In addition, recent structural data shows that coordination of pyruvate22 and reaction intermediate analogs23 to the CT domain induces significant active site rearrangements that promote carboxybiotin/BCCP domain binding in the active site. There is, however, a lack of structural or dynamic data showing a direct correlation between BCCP translocation and


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pyruvate binding in the CT domain. As such, the consequences of ligand occupancy and subsequent substrate-induced formation of a biotin-binding pocket in the CT domain on the dynamics of BCCP domain movement and global conformational changes/catalytic turnover are unclear. In order for the occupancy state of the CT domain to be the driving force behind the efficient coordination of catalytic events at the two spatially distinct active sites, pyruvate binding would not only have to induce BC domain product release/BCCP translocation, but also must remain coordinated to the active site long enough to react with the CO2 liberated from the incoming carboxybiotin intermediate24. If pyruvate dissociates prior to carboxybiotin arrival and subsequent decarboxylation, the resulting non-productive cleavage of MgATP would disconnect the activities of the two domains and decrease the overall catalytic efficency22. From numerous structural and kinetic4,

5, 7

studies, we know that catalysis occurs through an intermolecular

mechanism on a single face (Fig 1 B) but how or, even more fundamentally, which CT and BC domains are

8-10

communicating is not known. In order to identify the possible pathways by

which the CT domain communicates with the other domains in the PC tetramer, we aim to first determine if pyruvate binding in the CT domain stimulates the release of products from the BC domain on its own polypeptide chain (intramolecular) or the BC domain on a different polypeptide chain (intermolecular, Fig 2). To this end, we have generated functional, mixed hybrid tetramer forms of the Rhizobium etli PC holoenzyme from two different mutant forms of the enzyme (E218A and T882S; RePC numbering, Fig 3)5, 6. The E218A mutant has an inactive BC domain, a CT domain displaying reduced CT domain activity13 and, we anticipate, an apparent Km or Ka for pyruvate comparable to the wild-type enzyme. In contrast, the T882S mutant form of PC (characterized here) exhibits


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wild-type levels of BC domain activity and significantly reduced CT domain activity. In addition, a marked increase in the apparent Km for pyruvate contributes to the observed 100-fold decrease in kcat/Km pyruvate compared to the wild-type catalyzed carboxylation of pyruvate. Based on the predicted arrangement of monomers (Figs 4 and 5), we can distinguish between intra- and intermolecular control of BC domain product release by determining the kcat and apparent Km pyruvate

for the full forward reaction and the apparent Ka pyruvate for the stimulation of Pi release in

the BC domain. We report here the initial rates of Pi release and oxaloacetate formation as a function of increasing pyruvate concentrations for the wild-type, T882S mutant homotetramer, and mixed hybrid tetramer forms of PC. The observed kcat for majority of the hybrid tetramer catalyzed reactions were consistent with predicted values. More importantly, the apparent Ka pyruvate for the E218A:T882S[1:1] hybrid tetramer-catalyzed cleavage of MgATP and subsequent release of Pi was determined to be 0.29 ± 0.02 mM, comparable to the wild-type enzyme and nearly 10-fold lower than that determined for the T882S homotetramer. We also found that the coupling between the BC and CT domain reactions was incomplete when hybrid tetramers were generated using the T882S mutant (i.e. WT:T882S[1:1] and E218A:T882S[1:1]). The ratio of the rates of oxaloacetate formation to Pi release at all pyruvate concentrations for the WT:T882S[1:1] and E218A:T882S[1:1] catalyzed reactions was ~0.6, while the wild-type, WT:E218A[1:1] and T882S mutant homotetramer exhibited near 1:1 coupling between the BC and CT domain reactions. Our results are consistent with a mechanism where the occupancy of the CT domain governs BCCP domain translocation through an intermolecular mechanism and are instrumental in beginning to define the thermodynamic, kinetic and structural features of subunit communication pathways in PC.


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MATERIALS AND METHODS. Materials. IPTG, biotin, NADH, ampicillin, and chloramphenicol were purchased from Research Products International Corp. (RPI). 7-methyl-6-thioguanosine was obtained from Berry and Associates (Dexter, MI). Ni2+-Profinity IMAC resin was obtained from Bio-Rad. Pyruvate (sodium salt) was obtained from Fisher Scientific and the trilithium salt of acetyl-CoA was purchased from Crystal Chem (Downers Grove, IL). Malate dehydrogenase was purchased from Calzyme (San Luis Obispo, CA). All other reagents and coupling enzymes were obtained at the highest-grade purity from Sigma-Aldrich and used without further manipulation. Methods. Protein purification. The T882S and E218A mutant forms of RePC were previously generated using the QuikChange Site-Directed Mutagenesis Kit protocol (Agilent Technologies) and sequence verified

11, 13

. Recombinant wild-type and mutant forms of the enzyme were

overexpressed in Escherichia coli BL21 Star (DE3) and purified as previously described 7. Generation of functional, mixed hybrid tetramers. Concentrated stocks of wild-type PC and the T882S and E218A mutant forms were diluted with 100 mM Tricine (pH 7.6) to a final concentration of 1 mg/mL. Enzyme stocks were individually incubated at room temperature for 30 min to allow for complete dissociation of the tetramers into monomers

6, 25

. 1:1 hybrid

tetramers were generated by mixing equal volumes of the dilute enzyme stocks to a final concentration of 1 mg/mL (total protein). To facilitate the complete rehybridization of the monomers into a statistically random distribution of tetramers, the solutions were allowed to incubate an additional 30 min at room temperature in the presence of acetyl-CoA (0.24 mM final concentration5, 6). Similarly, the 4:1 and 1:4 T882S:E218A hybrid tetramers were generated by mixing the diluted T882S and E218A enzyme solutions in 4:1 and 1:4 ratios, respectively. We have adopted the use of a simplified naming convention when referring to the various hybrid


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tetramers where a colon separates the enzyme forms used and the bracketed subscript denotes the ratio of the enzyme stocks. For example, the hybrid tetramer generated from the incubation of the T882S and E218A mutants in a 1:4 ratio is denoted as T882S:E218A[1:4] HT. The hybrid enzymes were used without any further manipulation. Enzyme activity assays. The initial rates of oxaloacetate formation (full forward reaction) and Pi release (forward reaction of the BC domain) were determined spectrophotometrically using coupled assay systems. All reactions were performed at 25 °C in 1 mL reaction volumes at pH 7.6 (100 mM Tricine). Rates of pyruvate carboxylation were determined using the malate dehydrogenase coupled assay system and monitoring the concomitant oxidation of NADH to NAD+ at 340 nm (ε340 = 6220 M-1 cm-1)7. The initial rates of oxaloacetate formation were measured at varying concentrations of pyruvate (0.025 – 40 mM) and saturating concentrations of all other substrates and activators. Reactions contained 40 or 15 mM HCO3-, 2.5 mM MgATP, 2.5 mM MgCl2, 0.25 mM acetyl-CoA, 0.15 mM NADH and malate dehydrogenase (10-12 U). Reactions were initiated with the addition of ~1.5–800 µg of wild-type, mutant or hybrid tetramer forms of PC. Error bars are the standard deviation (SD) of three separate determinations. The initial rates of the HCO3--dependent cleavage of MgATP and subsequent release of Pi in the presence of varying concentrations of pyruvate (0-30 mM) were determined using the purine nucleoside phosphorylase (PNP) coupled assay system and monitoring the concomitant formation of 2-amino-6-mercapto-7-methylpurine from 7-methyl-6-thioguanosine (MESG) at 360 nm (ε360 = 6300 M-1 cm-1)

26, 27

. Reactions contained 40 mM HCO3-, 2.5 mM MgATP, 2.5

mM MgCl2, 0.25 mM acetyl-CoA, 0.2 mM MESG, and purine nucleoside phosphorylase (3-15


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U) and were initiated with ~1.5–350 µg of PC. Error bars are ± SD for three separate determinations. Data Analysis. Kinetic parameters (kcat and kcat/Km

pyruvate)

for pyruvate carboxylation were

determined by fitting initial velocity plots to the Michaelis-Menten equation (eqn 1) using nonlinear regression (Prism 7.0, GraphPad, San Diego CA)

vi =

k cat [pyruvate] (1) K m + [pyruvate]

where kcat (sec-1) is the rate of carboxylation at saturating pyruvate and Km (mM) is the apparent Michaelis-Menten constant for the full forward reaction. Errors reported for kinetic parameters were derived from the fit to eqn (1). kcat/Km values are determined from the ratio of the two parameters and error reported is the propagated error from the non-linear regression. Kinetic parameters (i.e. kcat and kcat/Ka

pyruvate)

for the pyruvate-stimulated release of Pi as a

function of pyruvate concentration were determined by fitting initial velocity plots to eqn (2)28

vi = k 0 +

k cat [pyruvate] (2) K a + [pyruvate]

where k0 (sec-1) is experimentally determined rate of Pi release in the absence of pyruvate, kcat (sec-1) is the rate of Pi release in the presence of saturating pyruvate and Ka is the apparent Michaelis-Menten constant for the stimulation of Pi release by pyruvate. To determine the extent of coupling between the BC and CT domain reactions, the ratios of the rates of oxaloacetate formation to the rates of Pi release were plotted as a function of increasing pyruvate concentrations. Data were fitted to a straight line and the error bars are the propagated ± standard deviations from three separate determinations. Probability analysis of monomer distribution after rehybridization and calculation of theoretical rates for intra- and intermolecular mechanisms. We have determined the probability


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of relative distribution of mutant or wild-type monomers for the homogenous population of the WT:E218A[1:1], WT:T882S[1:1], T882S:E218A[1:1], T882S:E218A[1:4] and T882S:E218A[4:1] hybrid tetramers. The rehybridization of two different PC monomers, for example the T882S and E218A mutant (Fig 4), gives rise to 4 different combinations of monomers on a single face (Fig 4A) and 16 unique tetramers (Fig 4B), regardless of the relative ratio of which the mutants were mixed. In order to determine the probability and relative contribution of any individual combination of monomers to the observed catalytic rate, we made several simplifying assumptions, namely that (1) all positions within the tetramer (i.e. M1, M2, M3, and M4) are equally and independently populated by either mutant, (2) the positioning of a monomer within the tetramer does not significantly reduce the remaining population of monomers in the solution (3) the activity of a monomer is essentially independent of the activity of surrounding monomers within the tetramer and (4) the monomers within a mixed, rehybridized heterotetramer exhibit similar activity and apparent Km or Ka values as the parent homotetramer. In particular, we made assumptions (3) and (4) to reduce the complexity of the calculations of the theoretical rates and will discuss the ramifications of these assumptions further in the Results and Discussion sections. For a mixture containing a 1:1 ratio of T882S and E218A monomers, the probability (p) that a T882S mutant monomer would fill any position (M1-M4) in the tetramer is given in eqn 3

= = = (3) where n and m are the relative ratios of T882S and E218A in solution. Similarly, the probability (q) of an E218A mutant monomer filling any position in the tetramer in a 1:1 mixture of mutants is given in eqn (4)

= = = (4)


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Because of the large number of T882S and E218A monomers in solution, these probabilities do not change when a monomer is positioned in the tetramer. Therefore, the probability (P) for forming any one of the 16 unique tetramers (Fig 4B) is determined by the eqn (5)

= (5) where k is the number of T882S monomers in the configuration (i.e. 0-4), p is the probability of the T882S filling any position in the tetramer (eqn 3) and q is the probability of any E218A monomer filling any position in the tetramer (eqn 4). For example, the probability of generating the hybrid tetramer combination with the T882S monomer in every position in a 1:1 mixture of T882S:E218A monomers (Fig 4B, combination 1) is given in eqn 6

= = = 0.0625 (6) while the probability for an E218A monomer occupying every position in a single tetramer (Fig 4B, combination 2) is

= = = 0.0625 (7) Similar reasoning can be used to determine the probability of distribution for a 1:4 or 4:1 mixture of T882S:E218A mutant monomers. For example, from eqn 3, the probability of positioning a T882S monomer in any position in the tetramer in a 1:4 mixture of T882S:E218A is

= = = (8) while the probability of positioning the E218A mutant in the same solution is

= = = (9) Combining eqns 8 and 9 with eqn 5, the probability of generating a mixed hybrid tetramer with all T882S monomers in a 1:4 mixture (Fig 4B, combination 1) is


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= =

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= 0.0016 (10)

Similarly, generating mixed hybrid tetramer with all E218A monomers in a 1:4 mixture (Fig 4B, combination 2) is

= =

= 0.41 (11)

To determine the contribution of a configuration to the overall observed rate of pyruvate carboxylation, we used the relative positioning of the monomers in the configuration to determine the unique intermolecular BC-CT domain interactions that would give rise to catalytic activity. There four possible catalytically relevant interactions within a single tetramer (i.e. BC1CT2, BC2-CT1, BC3-CT4 and BC4-CT3). In configurations 3-6 (Fig 4B), for example, each tetramer contains three T882S monomers and one E218A monomer, giving rise to two catalytic interactions between a T882S BC and CT domain (BCT882S-CTT882S), one T882S BC domain interaction with an E218A CT domain interaction (BCT882S-CTE218A) and one E218A BC domain interaction with a T882S CT domain (BCE218A-CTT882S). Based on the relative activities determined for the parent homotetramers, we assigned these interactions with relative % activity (i.e. BCT882S-CTT882S is 1.6% of wild-type activity, BCT882S-CTE218A is 3-5% of wild-type activity11, 13 and BCE218A- CTE218A is inactive). Moreover, because of the finite sample space and the independence of the individual configurations, the probability of any event is the sum of the probability of its elements. In this way, we can combine the probability of distribution with the relative rates of each interaction within the configuration to determine how much the configuration contributes to the overall kcat and determine predicted kcat values for pyruvate carboxylation for the 1:1, 1:4 and 4:1 T882S:E218A hybrid tetramers (Fig 4B). Similar probability analysis and predicted rates are given for the WT:E218A[1:1] and the WT:T882S[1:1] hybrid tetramers in the Supporting Information (SI Figs. 1 and 2, respectively).


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Essentially, the same methods were determined to calculate the predicted rates for the pyruvate stimulated release of Pi catalyzed by the T882S:E218A[1:1] (Fig 5), WT:T882S[1:1] (SI Fig 3) and WT:E218A[1:1] (SI Fig 4) hybrid tetramers. Probability distribution was determined as above, but in this case, the relative rate contribution was determined based on either an intramolecular or an intermolecular interaction. In the case of an intramolecular mechanism, the rate of Pi release from the active T882S BC domains in the presence of saturating concentrations of pyruvate will be solely controlled by the T882S CT domains on the same monomers. For example, in configurations 3-6 (Fig 5), three of the four PC monomers in the hybrid tetramer will exhibit activity consistent with the BC domain activity of the T882S holoenzyme (1.2% of wildtype activity), regardless of the identity or interactions with other monomers in the tetramer. The single E218A BC domain in this configuration would under the control of its own CT domain. In contrast, if we assume an intermolecular mechanism, that is pyruvate binding in the CT domain of an opposing polypeptide chain stimulates the BC domain reaction, we see that the calculated rate of Pi release for the same configuration is dependent on two T882S CT domain interactions (BCT882S-CTT882S, 1.2% of wild-type activity) and one E218A CT domain interaction (BCT882SCTE218A, 3-5% of wild-type activity) with the three catalytically active T882S BC domains. Again, since the events are independent, the probability and overall calculated kcat observed is the sum of all the events. It should be noted that the rate for the CT domain activity of the E218A monomer was based on the ability of the homotetramer to catalyze the oxamate-stimulated decarboxylation of oxaloacetate29, rather than pyruvate carboxylation, due to the inactive E218A BC domain. Because the origins of the decreased CT domain activity for the E218A mutant could have arisen from alternate kinetic pathways for decarboxylation29,

30

, half-the-sites reactivty31 and global


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conformational changes32, or possible unknown dominate negative effects propagated throughout the tetramer, we initially attributed the decreased CT domain activity to the inability to of the E218A homotetramer to promote oxaloacetate decarboxylation through an alternative kinetic pathway occurring in the BC domain29, 30. As such, we initially assumed the CT domain activity in an E218A monomer to exhibit 100% activity when paired with a fully active BC domain. In all calculations involving the E218A mutant, when 100% CTE218A activity was assumed, the predicted kcat values were significantly higher than the observed rates for both carboxylation and Pi release. In contrast, invoking an assumption where the CT domain active site exhibits activity that is 3-5% that of the wild-type enzyme, calculated rates were on par with what was observed for carboxylation and Pi release. We are unsure if the decreased activity is arising from intermolecular effects between the BC and CT domain on the E218A monomer or are a byproduct of complex regulatory mechanisms governing PC activity that we do not fully understand yet. That being said, based on the positioning of the mutation and recombination into hybrid tetramers, we can distinguish between an inter- and intramolecular control mechanism regarding Pi-release by relying heavily on the apparent Km and Ka data presented here. Further rationale and justification for this assumption in the context of an inter- and intramolecular control mechanism is presented in the Results and Discussion sections. RESULTS. Kinetic parameters for the PC catalyzed carboxylation of pyruvate to form oxaloacetate. In order to assess the overall pyruvate carboxylating abilities of the wild-type, mutant and mixed hybrid tetramer forms of PC, we determined the initial rates of oxaloacetate formation at varying concentrations of pyruvate (0.025-30 mM) and saturating concentrations of all other substrates and activators using the malate dehydrogenase coupled assay system (Table 1). All of the


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enzyme forms and hybrid tetramers exhibited a normal Michaelis-Menten type response to increasing pyruvate concentrations (Fig 6). In this instance, we expect that the apparent Km for pyruvate determined for oxaloacetate formation is a function of both the ability of pyruvate to stimulate Pi-release, possibly through triggering BCCP domain translocation, and catalytic turnover. In previous studies, we had determined kcat and kcat/Km for the wild-type PC catalyzed reaction at HCO3- concentrations near the apparent Km value (~10 mM)7. In the current study, we determined the same rates at both 15 and 40 mM HCO3- and observed a nearly 8-fold increase in kcat and concurrent 20-fold increase in the apparent kcat/Km (SI Fig 5). Taken together, the significant kcat and kcat/Km pyruvate effect observed with increasing HCO3- concentrations suggest that sub-saturating concentrations of the substrate used in our previous study may have artificially inflated the apparent Km pyruvate. We will discuss the ramifications of this decrease in regards to reaction coupling between the spatially distinct active sites in subsequent sections but have modified the experimental design in this study such that experiments were performed in the presence of saturating (40 mM) concentrations of HCO3-. Demonstrated in previous studies10,

11

, the strictly conserved Thr882 residue is essential to

pyruvate carboxylation and proposed to be responsible for proton shuttling between the biotin enolate and bound pyruvate in the CT domain. As shown in Table 1, a conservative Thr to Ser mutation had devastating effects on the overall catalytic activity. The T882S mutant PC homotetramer exhibited a 62-fold decrease in activity and 360-fold decrease in kcat/Km compared to wild-type PC. More importantly, the low CT domain activity and high apparent Km for pyruvate (Km = 1.3 ± 0.2 mM) exhibited by the T882S mutant form of PC made it ideal for generating the mixed hybrid PC tetramers.


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The incorporation of the E218A mutation results in an enzyme with a completely inactive BC domain and, we expect, an apparent Km

pyruvate

comparable to the wild-type enzyme and a CT

domain with reduced activity (3-5%) as measured by the ability of the E218A homotetramer to catalyze the oxamate-induced decarboxylation of oxaloacetate13. The decreased CT domain activity could be due to (1) the inactive E218A BC domain influencing the ability of its own CT domain to catalyze carboxylation/decarboxylation, (2) the inability of the E21A homotetramer to catalyze the decarboxylation of oxaloacetate through an alternative kinetic pathway involving the BC domain29, 30 or (3) difficulty in adopting global or local conformational changes that aid in facilitating catalysis as part of a more complex regulatory mechanism. In fact, the reduced activity is most likely a combination of these three mechanisms. Because of the numerous catalytic, dynamic and regulatory steps occurring in PC, it is difficult from the kcat data alone to determine exactly why the E218A CT domain exhibits reduced in activity. We first generated functional, mixed hybrid tetramers from the wild-type and mutant forms of the enzyme. The WT:T882S[1:1] hybrid displayed lower than predicted carboxylation activity (Table 1 kcat

calc,

SI Fig 1), that is 31% of wild-type activity vs. 51%, respectively, but,

surprisingly exhibited near wild-type apparent Km for pyruvate but . In order to confirm that the reduced rate was not a byproduct of the rehybridization method, we measured the specific activities of the WT:E218A[1:1] hybrid tetramer at saturating concentrations of pyruvate (40 mM). The observed rates were ~3-fold lower than the wild-type enzyme and near the predicted values (SI Fig 2). While the kcat data alone could be interpreted as incomplete rehybridization, the apparent Km data described below and the initial rates determined for Pi-release indicates that the incorporation of either the CT or BC domain mutation did not affect the ability of the monomers to rehybridize in a statistically random manner. We suspect, based on data presented in the


16

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Biochemistry

following sections, that the differences in the observed and predicted rates arises from the reduced ability of the T882S monomers in the hybrid tetramers to effectively control coupling between the BC and CT domains. Similar to the WT:T882S[1:1] hybrid tetramer, the T882S:E218A[1:1] hybrid tetramer exhibited 50% of the predicted activity (Figs 4 and 7) and, more importantly, an ~5-fold decrease in the apparent Km for pyruvate as compared to the T882S homotetramer. In order to make sure that incorporation of the T882S mutation did not alter the ability of the enzyme to tetramerize, we determined the carboxylating ability of hybrid tetramers generated from mixing different ratios of the two mutant forms of the enzyme (Fig 7). The observed carboxylation activity of the T882S:E218A[4:1] hybrid tetramer was the same as the predicted rate (Fig 4) and the 4-fold increase in the apparent Km

pyruvate

as compared to the wild-type enzyme is reflective of the

relative increase in T882S monomers incorporated into the tetramers. Interestingly, generating hybrid tetramers with an excess of the E218A mutant monomer resulted in enzymes with observed activity twice that of the predicted values, even though the apparent Km for pyruvate is near that determined for the T882S homotetramer (Figs 4 and 7). More importantly, we would expect to see drastically different apparent Km values when the tetramers were generated using different ratios had the monomers not rehybridized in statistically random populations. Specific activities for MgATP-cleavage/Pi release in the absence of pyruvate. Strictly occurring in the BC domain, MgATP cleavage and Pi release occurs in the absence of pyruvate, albeit at a significantly reduced rate. The rate of Pi release is proposed to be dependent on the BCCP domain movement away from the BC domain where it is positioned directly over the Bsubdomain lid6, 7, 13. Translocation of the BCCP domain presumably allows the B-subdomain/BC domain to adopt a more open conformation that facilitates product release. Pyruvate binding in


17

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Page 18 of 49

the CT domain is thought to trigger the translocation of the BCCP domain19, 20 and significantly increases the rate of Pi release33, however, in the absence of the substrate, dynamic fluctuations of the enzyme allow for the random movement of the BCCP domain to the CT domain, albeit at a significantly reduced rate/amount. Specific activities for Pi release in the absence of pyruvate were determined for the mutant homotetramers and hybrid tetramers (Table 2). The activity of the T882S homotetramer was 17-fold less than wild-type while the E218A homotetramer was completely inactive due to the mutation in the BC domain. Because half of the BC domain active sites in the WT:E218A[1:1] hybrid tetramer are inactive, we expected to observe a 50% reduction in the rate of Pi release compared to wild-type. While the observed rate was significantly less than predicted (34% of wild-type), this type of dominant negative effect has been observed in acetyl-CoA carboxylase34 and is attributed to a currently undefined cooperative communication mechanism. The WT:T882S[1:1] hybrid tetramer was more active than the T882S homotetramer and ~50% as active as the wild-type enzyme. Not surprisingly, the T882S:E218A[1:1] hybrid tetramer exhibited a 3-fold decrease in activity compared to the T882S mutant form of the enzyme, consistent with the pattern of activity observed in the WT:E218A[1:1] hybrid tetramer. Pyruvate-stimulated release of Pi. In the presence of pyruvate, the substrate-induced translocation of the BCCP domain away from the BC domain to the CT domain on an opposing polypeptide chain significantly increases the rate of Pi release from the BC domain16, 18, 33. The initial rates of Pi release were determined as a function of increasing pyruvate concentration (Fig 6, Table 3) and fixed saturating concentrations of all other substrates and activators (i.e. 40 mM HCO3-). To determine the kinetic parameters for the reaction, data were fitted to a modified Michaelis-Menten equation (eqn 2) where the experimentally determined rate of Pi release in the


18

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Biochemistry

absence of pyruvate was accounted for. Since we are measuring the initial rates of the reaction in the BC domain and the increase in the rate of Pi release in the presence of pyruvate arises from the substrate-induced translocation of the BCCP domain, the apparent Ka for pyruvate is a measure of the ability of pyruvate to stimulate the release of products from the BC domain and is not reflective of complete catalytic turnover, nor is it necessarily predicted to be the same as the apparent Km for carboxylation. In general, the presence of pyruvate stimulated the rate of Pi release (3.5-20-fold) and, with the exception of the T882S:E218A[1:1] hybrid tetramer, increased the apparent Ka for pyruvate as compared to the apparent Km

pyruvate

for the full forward reaction. The presence of saturating

pyruvate (kcat) resulted in a 16.5-fold increase in the wild-type catalyzed rates of Pi release as compared to in its absence. In contrast to our previous results7, we observe a nearly 2-fold increase in the apparent Ka for pyruvate compared to the apparent Km determined for the full forward reaction. We attribute these differences to the sub-saturating concentrations of HCO3used in the previous studies as described above. The rate of the T882S homotetramer catalyzed release of Pi in the presence of saturating pyruvate was 3.4-fold greater than that determined in its absence. The apparent Ka was ~4-fold greater than the apparent Km for pyruvate carboxylation and 10-fold greater than that for wildtype. The increased Ka and significant decrease in kcat results in a nearly 780-fold reduction in kcat/Ka, compared to wild-type, suggesting that the T882S mutation significantly hinders the ability of the pyruvate to stimulate the release of Pi from the BC domain. In order to differentiate which CT domain, that is on the same monomer or different monomer, controls the movement of the BCCP domain, we next determined the rates of Pi release as a function of varying pyruvate for wild-type:mutant and double mutant hybrid tetramers.


19

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Page 20 of 49

Comparing the experimentally observed rates with the predicted rates for an intra- or intermolecular mechanism, we can determine which mechanism predominates during turnover by measuring the overall influence of pyruvate on the rate of Pi release. The WT:T882S[1:1] hybrid tetramer exhibited a 20-fold increase in the rate of Pi release as compared to the nonpyruvate stimulated rate. Based on our probability analysis (SI Fig 3), the predicted rate for either the intra- or an intermolecularly controlled movement of the BCCP domain using the WT:T882S[1:1] would be ~51% of wild-type activity (Table 3, kcat

inter calc

and kcat

intra calc,

respectively). In contrast to our results for pyruvate carboxylation, the observed rate of pyruvatestimulated Pi release (~55% of wild-type activity) is near the predicted values. These results suggest that differences between the calculated and observed rates for the WT:T882S[1:1] hybrid tetramer catalyzed carboxylation of pyruvate are most likely arising from incomplete coupling between the BC and CT domain reactions13, rather than incomplete rehybridization. The WT:T882S[1:1] hybrid tetramer showed a 2-fold increase in the apparent Ka for pyruvate compared to the Km pyruvate for carboxylation. Interestingly, the Ka

pyruvate

for the mixed hybrid

tetramer is slightly greater than the wild-type enzyme (1.6-fold increase) however, it is still significantly less than the T882S homotetramer (6-fold decrease). Saturating concentrations of pyruvate (40 mM) stimulated the rate of the WT:E218A[1:1] hybrid tetramer catalyzed release of Pi ~15-fold. From the unique arrangement of the individual monomers in the WT:E218A[1:1] hybrid tetramer (SI Fig 4), we can see that any Pi release activity arises from the fully active wild-type BC domain. In the case of an intramolecular mechanism, pyruvate binding to the wild-type CT domain on the same stimulates Pi release, resulting in an enzyme exhibiting 50% of the wild-type activity (Table 3). In contrast, in an intermolecular mechanism pyruvate binding in either a wild-type or E218A CT domain could


20

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Biochemistry

influence wild-type BC domain activity depending on the relative arrangement of the monomers. Because of the reduced activity of the E218A CT domain, determined in previous studies to be 3-5% of the wild-type catalyzed reaction13, the predicted rate of Pi release is ~26% of wild-type activity. The specific activity of the WT:E218A[1:1] catalyzed release of Pi at saturating concentrations of pyruvate (40 mM) was ~ 3-fold lower than the wild-type activity (31% of wildtype) and consistent with an intermolecular mechanism. To further support the intermolecular nature of the mechanism governing the substrate-induced stimulation of Pi release from the BC domain, we determined the initial rates of the T882S:E218A[1:1] hybrid tetramer catalyzed release of Pi as a function of varying pyruvate concentration. In an intramolecular mechanism, Pi release would be stimulated solely by pyruvate binding in the CT domain active site of the same monomer. For the T882S:E218A[1:1] hybrid tetramer, we would expect an apparent Ka pyruvate on par with the T882S homotetramer (~5 mM) since Pi release is only occurring in the active T882S BC domains under the control of the CT domain on the same monomer. In contrast, if the mechanism were intermolecular, then Pi release is governed by pyruvate binding to the CT domain on a different polypeptide chain. In this way, depending on the relative arrangements of the monomers on a face of the tetramer, pyruvate binding to either T882S and E218A CT domains on opposing polypeptide chains could feasibly trigger translocation, giving rise to apparent Ka pyruvate on par with the wild-type enzyme. Similar to the WT:E218A[1:1] hybrid tetramer, we can predict the rate of pyruvate-stimulated Pi release for the T882S:E218A[1:1] catalyzed reaction relative to the wild-type enzyme under the control of an intra- or intermolecular mechanism (Fig 5). While the difference in the predicted rates between the inter- and intramolecular mechanism are small (0.9 % vs. 1.1 %), the expected differences in the apparent Ka pyruvate are large enough to support the existence of one mechanism


21

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Page 22 of 49

over another. The T882S:E218A[1:1] hybrid tetramer was nearly as active as the T882S homotetramer (1.2-fold decrease) and 100-fold less active than the wild-type enzyme. Most notable and important to establishing the communication mechanism, though, was the 15-fold decrease in the apparent Ka for pyruvate compared to the T882S homotetramer. In fact, the apparent Ka was 1.5-fold lower than that observed for the wild-type enzyme. Taken together, these results show an increased ability of pyruvate to stimulate the release of Pi in the T882S:E218A[1:1] hybrid tetramer compared to the T882S homotetramer, as evidenced by the nearly 12-fold increase in kcat/Ka. In addition, these results are consistent with an intermolecular control mechanism, where pyruvate binding to the E218A CT domain, which is expected to exhibit comparable wild-type like affinity for pyruvate, on a different polypeptide chain significantly contributes to increasing the rate of Pi release by triggering the translocation of T882S BCCP domain on a different polypeptide chains. If the T882S CT domain governs the movements of its own BCCP domain (Fig 5), we would expect to have observed Ka values on par with the T882S homotetramer. Instead, the significantly reduced Ka, (Table 3) indicates that pyruvate binding to the CT domain of one polypeptide chain stimulates the rate of Pi release from a BC domain on a different polypeptide chain. Coupling between the BC and CT domain Reactions. The ratio of the rates of oxaloacetate formation to Pi release as varying concentrations of pyruvate is a measure of the relative coupling between MgATP-cleavage/Pi release in the BC domain with oxaloacetate formation in the CT domain (Fig 8). Ratios near one indicate complete coupling between the cleavage of MgATP and oxaloacetate formation while ratios below one indicate incomplete coupling and non-productive MgATP cleavage. Comparison of the kcat for oxaloacetate formation to the kcat


22

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Biochemistry

for Pi release show that, at saturating concentrations of pyruvate, the wild-type, T882S homotetramer and WT:E218A[1:1] hybrid tetramer exhibit nearly complete coupling between the reactions of the two domains. In contrast, mixed hybrid tetramers formed with the T882S mutant monomers consistently exhibit ratios of kcat oxaloacetate formation to kcat Pi release that are less than one (~0.5-0.6), indicating a significant degree of non-productive MgATP cleavage. These results were somewhat surprising considering that the T882S homotetramer shows complete coupling at all concentrations of pyruvate (Fig 8). Interestingly, we observed a linear dependence of the ratio of the rates on increasing concentrations of pyruvate for the wild-type, T882S homotetramer and mixed hybrid tetramers. The linear dependence of BC-CT domain coupling on pyruvate was unexpected considering the hyperbolic dependence we observed in our previous studies7,

13

. Again, we attribute the

discrepancy between the two studies to the sub-saturating concentrations of HCO3- used previously, which, as shown here, has a marked effect on the apparent Km for pyruvate. The ratio of kcat/Km pyruvate to kcat/Ka pyruvate for the T882S homotetramer (~5) was 2-fold greater than the wild-type enzyme (~2), suggesting that the mutation had a greater effect on pyruvate binding and the ability of the CT domain to facilitate Pi release than on overall turnover. This is also apparent in the nearly 5-fold increase in the apparent Ka pyruvate for the T882S homotetramer compared to the apparent Km for the overall reaction. In contrast, the wild-type enzyme exhibits a more modest 2.5-fold increase. The mixed hybrid tetramers generated with the T882S mutant also have a lower kcat/Km

pyruvate

to kcat/Ka

pyruvate

ratio than the T822S homotetramer, most likely

because the wild-type and wild-type-like CT domain of the E218A mutant significantly enhances the ability of the enzyme to facilitate Pi release in the BC domain (Table 4).


23

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Page 24 of 49

Taken together, our results show that (1) pyruvate binding in the CT domain controls the movement of a BCCP domain on a different polypeptide chain via an intermolecular mechanism and (2) mixed hybrid tetramers generated with the T882S CT domain exhibit a loss of coupling between the BC and CT domain reactions. In addition, our results may indicate that half-of-thesites reactivity and/or cooperative effects among the four monomers may be important contributors to the catalytic mechanism of the PC holoenzyme.

DISCUSSION. The ability of multifunctional enzymes to coordinate catalytic events occurring at spatially distinct active sites is imperative to efficient catalytic turnover and, in the case of PC, metabolic homeostasis 3. Previous steady-state kinetic studies

7, 17, 18, 35

indicate that, in the presence of

acetyl-CoA, pyruvate binding in the CT domain stimulates the release of Pi from the BC domain 7, 13, 36

, possibly through facilitating the translocation of the BCCP domain6, and increases the

amount of productive MgATP cleavage in the BC domain33. Collectively, these results suggest that the ability of pyruvate to coordinate spatially discrete catalytic events in PC may hinge on its ability to induce the intermolecular translocation of the BCCP domain and remain bound to the CT domain active site long enough to react. Interestingly, the available structural data suggests that pyruvate binding in the CT domain active site does not alter the overall global conformation of the enzyme 22, 23, 32, 37, but does induce significant structural alterations within and around the CT domain active site 22, 23. The pyruvate induced conformational remodeling of the active site is presumably a key driving force in the coordination of catalytic events, considering the conformational rearrangements of the CT domain result in the formation of the imperative biotin-binding pocket.


24

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Biochemistry

The influence of pyruvate occupancy of the CT domain active site on BC domain activity has led us to question whether pyruvate binding to the CT domain facilitates BC domain product release through an intra- or intersubunit communication mechanism (Fig 2B). Here, we report our first efforts in teasing apart the complex mechanisms governing catalytic coordination in PC by determining whether the “signal” for is originating from the CT domain on the same or neighboring polypeptide chain. By using mixed functional hybrid tetramers generated from strategically chosen mutant forms of PC, we have designed a system that will allow us to distinguish between intra- or intersubunit control (Fig 2). Based on the predicted arrangement of mutant PC monomers within the rehybridized tetramers (Figs 4 and 5), we can distinguish between an intra- and intermolecular mechanism by determining the kcat and, more importantly, the apparent Km pyruvate for the full forward reaction and the apparent Ka pyruvate for the stimulation of Pi release in the BC domain. Specifically, if the T882S CT domain, which exhibits a high apparent Ka for pyruvate relative to the wild-type like CT domain of the E218A mutant, stimulates product release (i.e. Pi and MgADP) from the BC domain through intramolecular interactions within a single subunit, we expect to observe an increased Ka pyruvate for Pi release compared to the wild-type enzyme (Fig 2B). In contrast, if pyruvate binding to the wild-type CT domain of the E218A mutant governs the translocation of the BCCP domain through intermolecular interactions between subunits, we expect that the resulting Ka

pyruvate

will be

significantly lower than that determined for the T882S mutant homotetramer (Fig 5). From our current steady-state kinetic analysis of the functional, mixed hybrid tetramers, we have determined that pyruvate binding to the CT domain active site facilitates the release of Pi from a BC domain on a different polypeptide chain (intermolecular/intersubunit communication mechanism). Considering the lack of significant contacts between the opposing BC and CT


25

Biochemistry

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domains on a single face5,

8, 10

Page 26 of 49

, our data may be explained by an intersubunit communication

mechanism that is dominated by energetic (i.e. thermodynamics and conformational selection)3841

instead of structural (i.e. physical interactions of protein residue networks)42, 43 phenomena.

We can envision a thermodynamically driven intersubunit communication mechanism where the enthalpy and/or entropy associated with pyruvate binding, conformational remodeling of active sites or the stabilization of conformational ensembles is the “signal” of CT domain occupancy sensed by the BCCP/BC domain on a different polypeptide chain38-41, 44, 45. An alternative mechanism to also consider is one where the signal for translocation is coming from the CT domain on a polypeptide chain residing on a different face. While we have made the simplifying assumption throughout our discussion of probability distribution that the intersubunit communication is occurring between monomers on a single face, it is plausible that binding of pyruvate to the CT domain active site controls the translocation of the BCCP domain on the opposing face via the propagation of conformational changes through protein residue networks42, 46-48

between the extensive CTTOP-CTBOTTOM interface.

The data presented here cannot distinguish between intersubunit interactions across or between faces, only that product release from the BC domain is stimulated by a CT domain on a different polypeptide chain. A communication mechanism controlling the movement of the BCCP domain on a monomer positioned in the opposing face as a function of relative substrate binding is reminiscent of a half-the-sites reactive or obligatory oscillating catalytic mechanism where only one face of the tetramer is active as a time9, 32 and substrate binding/product release from one subunit influences the activity of the other face21, 49. A half-the-sites mechanism, where subunit interactions either across or between the faces, could account for the lower than calculated rates we observed with some of the hybrid tetramers. While the subunits were treated as independent


26

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Biochemistry

monomers in our calculations, it could be that unaccounted for subunit interactions are detrimental to the coordination of catalysis and only become apparent when mutated monomers are mixed in the hybrid tetramers with monomers that display an unaltered ability to interact with other subunits. The half-the-sites reactive mechanism may also help explain the incomplete coupling observed between the BC and CT domains of hybrid tetramers generated with the T882S mutant monomers. In these hybrid tetramers, both the T882S CT and E218A CT domains contribute to the stimulation of Pi release within the homogenous mixture of tetramers. The contribution of both these CT domains to Pi-release may give rise to incomplete coupling because of significant differences in catalytic turnover rates and relative pyruvate binding efficiency and/or turnover. For example, we observe incomplete coupling of BC and CT domain reactions in hybrid tetramers generated with the T882S monomer, but not in the T882S homotetramer where all the subunits are operating under the same “reduced” capacity to induce regulation/turnover in a halfthe-sites reactive mechanism. While purely speculative, our kcat data does bring up intriguing possibilities that may be consistent with a more complex regulatory mechanism that may involve the global asymmetric-to-symmetric transitions observed in recent Cryo-EM studies9 and halfthe-sites reactivity. An alternative explanation may be that PC does not exhibit half-the-sites reactivity and that interactions between subunits on a face merely results in a dominant negative effect on catalysis that propagated through non-descript intersubunit interactions. At this time, we are unsure whether the incomplete coupling between the BC and CT domain reactions in hybrid tetramers generated with the T882S mutant is a direct reflection of top/bottom face communication or a consequence of dominant negative kinetic effects propagated throughout the tetramer7, 13, 22, 23. Even so, based on previous kinetic and structural data, both a structurally or


27

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Page 28 of 49

thermodynamically driven intersubunit communication mechanism is equally probable. Communication between CT and BC domains on different polypeptide chains most likely occurs through a mechanism that is thermodynamically, structurally and kinetically driven38 and continuing experiments in our lab aim to determine the exact nature of the intermolecular communication pathway.

CONCLUSION. In order to address how CT domain ligand occupancy is “sensed” by other domains within the PC tetramer, we generated functional hybrid tetramers using the E218A (inactive BC domain) and T882S (low CT domain activity, high apparent Km /Ka

pyruvate)

mutant forms of PC. The

apparent Ka pyruvate for the pyruvate stimulated release of Pi catalyzed by the E218A:T882S[1:1] hybrid tetramer was comparable to the wild-type enzyme and nearly 10-fold lower than that determined for the T882S mutant. In addition, the ratio of the rates of oxaloacetate formation to Pi release at all pyruvate concentrations for the WT:T882S[1:1] and E218A:T882S[1:1] catalyzed reactions was 0.5 and 0.6, respectively. Collectively, these data are consistent with a mechanism where pyruvate binding to the CT domain increases rate of product release (Pi and MgADP) from the BC domain on a different polypeptide chain, suggesting that allostery inters/intrasubunit communication in PC may be driven, in part, by thermodynamics conformation stability.


28

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Biochemistry

FIGURES

Figure 1. Overall structural arrangement and general reaction scheme for PC. (A) Top: Cartoon representation of the crystallographic structure of a single PC monomer from Rhizobium etli (pdb ID 2QF75). Each polypeptide chain contains a biotin carboxylase domain (BC domain, blue), an allosteric domain (green), carboxyl transferase domain (CT domain, yellow) and biotin carboxyl carrier protein domain (BCCP domain, red). Biotin is covalently attached to a strictly conserved Lys residue in the BCCP domain (Lys1119, RePC numbering). MgATP-γ-S and ethylCoA are shown as sticks bound in the BC and allosteric domains, respectively. Bottom: Simplified 2D representation of the PC monomer as squares (BC domain), circles (CT domain) and ovals (BCCP domain). The color of the domains is consistent with that in the crystal structure representation. (B) Left: The overall architecture of the PC tetrameric holoenzyme. Monomers on a single face (M1 and M2, outlined in black) are positioned antiparallel relative to each other. The two faces, arbitrarily designated top (M1 and M2) and bottom (M3 and M4), are positioned perpendicularly to each other such that the tetramer is stabilized at each corner


29

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Page 30 of 49

through significant BCTOP-BCBOTTOM and CTTOP-CTBOTTOM residue contacts. Catalysis occurs through an intermolecular mechanism (green arrow) where the BCCP domain swings from the BC domain on the same monomer (i.e. M1) to the CT domain in an opposing polypeptide chain (M2) on the same face5-7. Right: Simplified 2-D representation of the PC holoenzyme. Green arrows indicate potential catalytically productive intermolecular interactions (i.e. BC1-CT2, BC3CT4). (C) Pyruvate carboxylation occurs in two distinct steps at the two spatially discrete active sites. (1) Biotin, which is covalently tethered to the BCCP domain, is initially carboxylated in the BC domain through the ATP-dependent generation of CO2 from HCO3-. Once carboxylated, the BCCP domain translocates to the CT domain on an opposing polypeptide chain (2) where the carboxybiotin intermediate is decarboxylated and pyruvate is carboxylated to generate oxaloacetate. The presence of pyruvate in the CT domain stimulates the release of Pi from the BC domain in reaction step 1 by facilitating the movement of the BCCP domain away from the B-subdomain lid of the BC domain and allowing for unrestricted product release from the BC domain6, System

18, 19

. All crystal structure figures were generated using PyMOL Molecular Graphics (v1.8,

Schrödinger,

LLC).


30

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Biochemistry

Figure 2. Pyruvate stimulates the release of Pi and movement of the BCCP domain through an intramolecular (A) or intermolecular (B) mechanism. In an intramolecular mechanism, binding of pyruvate in the CT domain of monomer 1 (CT1) would trigger the translocation of the BCCP domain on the same monomer (BCCP1) to the CT domain on an opposing polypeptide chain (CT2) where catalysis occurs. In an intermolecular mechanism, binding of pyruvate to the CT domain (CT2) would trigger the movement of the BCCP domain on the opposite chain (BCCP1). In this mechanism, the signal for translocation and catalysis would occur in the same CT domain. The extent of pyruvate’s effectiveness in promoting BCCP translocation is apparent in the increase in the rate of Pi release from the BC domain. In either mechanism, different apparent Km pyruvate for catalysis and apparent Ka

pyruvate

for the stimulation of Pi release/BCCP

translocation is assumed. Green arrows indicate the movement of the BCCP domain in relation to the CT domain controlling the translocation. For simplicity, only one of the possibly two catalytically active translocations (i.e. BC1 to CT2) is shown.


31

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Page 32 of 49

Figure 3. Representation of possible catalytic interactions between the T882S and E218A monomers after rehybridization into functional tetramers. (A) The T882S mutant form of PC has an active BC domain (blue square) and a CT domain (white circles) with reduced activity and increased apparent Km pyruvate compared to the WT enzyme. The E218A mutations rendered the BC domain completely inactive (light blue squares) and a CT domain exhibiting activity 35% of the WT enzyme but comparable Km for pyruvate (orange circles). (B) Upon rehybridization, a tetrameric face with two E218A monomers will be completely inactive while two T882S monomers on a single face will display low activity relative to the WT enzyme. A combination of T882S and E218A monomers on a single face will have one completely inactive BC-CT domain interaction, due to the inactive E218A mutant BC domain, and an active interaction between the T882S BC domain and E218A CT domain. Based on this arrangement, if pyruvate binding stimulates the rate of Pi release through an intramolecular mechanism, we expect to observe an apparent Ka significantly higher than the WT enzyme, due to control arising from the low activity/high apparent Ka pyruvate T882S CT domain (white circle). In contrast, if the mechanism is intermolecular, we expect to observe an apparent Ka on par with WT values due to control arising from pyruvate binding in the low activity CT domain of the E218A mutant which has

an

apparent

Km

on

par

with

the

wild-type

enzyme

(orange

circle).


32

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Figure 4. Probability of distribution and predicted pyruvate carboxylation activity for the rehybridized PC tetramers generated from mixing the T882S and E218A mutants in a 1:1, 4:1 and 1:4 ratio. (A)

Rehybridization of the T882S and E218A mutant monomers of PC

gives rise to 4 unique combinations on a single face of the tetramer (position M1 or M2). Each face has two possible intermolecular, catalytically active interactions, namely between the BC domain of monomer 1 and CT domain of monomer two (BC1 and CT2, respectively) and the BC CT domain interaction between monomer 2 and monomer 1 (BC2-CT1). Faces containing two T882S or two E218A monomers are predicted to exhibit 1.6% and 0% pyruvate carboxylating ability as compared to the wild-type enzyme. A combination of T882S and E218A monomers on a single face, regardless of the relative positioning in M1 or M2, will have a single inactive catalytic interaction between the BC domain of the E218A mutant and CT domain of T882S and a low activity (3-5% of wild-type), high pyruvate affinity catalytic interaction owing to the interaction between the T882S BC domain and E218A CT domain. (B) The random and stochastic rehybridization of the two monomers gives rise to 16 unique tetramer combinations. The probability of each configuration (rounded brackets) and overall catalytic contribution (square brackets) were determined based on the relative ratio of mixing and described in detail in the data analysis section. Predicted pyruvate carboxylation activity is determined from the sum of

the

individual

activity

and

probability

for

each

tetrameric

configuration.


34

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Figure 5. Predicted activities for the intramolecularly or intermolecularly controlled pyruvate stimulated release of Pi catalyzed by the T882S:E218A[1:1] hybrid tetramer. Similar to Fig 4, the relative distribution and probability for the 16 unique combinations of the hybrid tetramers (round brackets). For the intramolecular mechanism, the rate of Pi release is assumed to be under the control of the CT domain on the same monomer, i.e. CT1 controls BC1. In this instance, the overall rate of Pi release at saturating pyruvate would be solely due to the T882S monomers in the tetramer since the E218A BC domain is inactive. In the intermolecular


35

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control mechanism, the rate of Pi release is assumed to be influenced by the CT domain on an opposing polypeptide chain, i.e. CT1 influences activity in BC2. In this way, the CT domain of the E218A mutant will have an effect on activity and contribute to the overall catalytic rate when it is paired with a T882S BC domain. The determination of probability distribution and activity is further

detailed

in

the

data

analysis

section.


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Biochemistry

Figure 6. Initial velocity plots of the rate of oxaloacetate formation (black) and Pi release (red) as a function of increasing [pyruvate] for reactions catalyzed by the WT (A), T882S (B), WT:T882S[1:1] hybrid tetramer (C) and T882S:E218A[1:1] hybrid tetramer forms of the enzyme. Dashed lines are the best-fit line to either the Michaelis-Menten equation (black, eqn 1) or modified Michaelis-Menten equation (red, eqn 2). Error bars are ± standard deviation from three separate determinations. All data points were used for non-linear regression analysis.


37

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Figure 7. Initial velocity plots for the rate of oxaloacetate formation catalyzed by the T882S:E218A[1:1] (black), T882S:E218A[1:4] (blue) and T882S:E218A[4:1] (red) hybrid tetramers. Dashed lines are best-fit lines to the Michaelis-Menten equation (eqn 1) and error bars are ± standard deviation from three separate determinations. All data points were used for non-linear regression analysis.


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Biochemistry

Figure 8. Ratio of the rates of oxaloacetate formation to Pi release as a function of increasing pyruvate concentrations for the T882S (red) and T882S:E218A[1:1] (blue) hybrid tetramer catalyzed reactions. Data were fitted to a straight line using linear regression and error

bars

are

±

the

propagated

standard

deviations

determined

for

each

rate.


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TABLES. TABLE 1. Kinetic parameters for the PC-catalyzed carboxylation of pyruvate to form oxaloacetatea. kcat obs

Km pyruvate

kcat/Km

(sec-1)c

% WT Activity(calc)

(mM)d

(s-1 mM-1)

---

---

--

0.54 ± 0.08

2.3 ± 0.4

10.3 ± 0.2

(100)

---

(100)

0.22 ± 0.03

47 ± 6

T882S

0.166 ± 0.005

1.6

---

---

1.3 ± 0.2

0.13 ± 0.02

E218A

NAe

0

---

0

---

---

WT:T882S[1:1] HT

3.26 ± 0.06

31

5.2 ± 0.1

51

0.33 ± 0.03

9.9 ± 0.9

WT:E218A[1:1] HTf

3.23 ± 0.01

31

2.68 ± 0.05

26

NDg

ND

T882S:E218A[1:1] HT

0.075 ± 0.002

0.7

0.143 ± 0.003

1.4

0.20 ± 0.03

0.38 ± 0.06

T882S:E218A[4:1] HT

0.165 ± 0.004

1.6

0.152 ± 0.003

1.5

0.8 ± 0.1

0.21 ± 0.03

T882S:E218A[1:4] HT

0.121 ± 0.006

1.2

0.058 ± 0.001

0.6

1.6 ± 0.3

0.081 ± 0.001

WT

kcat calculated

(sec-1)b

% WT Activity(obs)

15 mM HCO3-

1.25 ± 0.04

40 mM HCO3-

Reaction conditions: Tricine (100 mM, pH 7.5, 25 °C), HCO3- (40 mM), pyruvate (0.025-30 mM), MgATP (2.5 mM), MgCl2 (2.5 mM), acetyl-CoA (0.25 mM), NADH (0.15 mM), malate dehydrogenase (10 U). bInitial velocities at each concentration were determined in triplicate. Kinetic parameters are derived from fits of all data points to the Michaelis-Menten equation (eqn 1) using non-linear regression. Errors reported are the standard deviation determined from the best fit to eqn 1. cPredicted activity based on the probability of tetramer arrangements and relative activity of monomers within the tetramer as described in the methods section. d Apparent Km. eNo activity detected. f Specific activities for the WT:E218A[1:1] HT were determined in triplicate at 40 mM of pyruvate and saturating concentrations of all other substrates and activators. g Not determined. a


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Biochemistry

TABLE 2. Specific activities for the PC-catalyzed release of Pi in the absence of pyruvatea. kcat obsb

% WT

(sec-1)

Activity

0.73 ± 0.09

(100)

T882S

0.043 ± 0.006

6

E218A

NAc

0

WT:E218A[1:1] HT

0.249 ± 0.009

34

WT:T882S[1:1] HT

0.33 ± 0.07

45

0.0134 ± 0.002

2

WT

T882S:E218A[1:1] HT

Reaction conditions: Tricine (100 mM, pH 7.5, 25 °C), HCO3- (40 mM), MgATP (2.5 mM), MgCl2 (2.5 mM), acetyl-CoA (0.25 mM), MESG (0.2 mM), PNP (3-15 U). b Specific activities determined in triplicate. Reported error is ± standard deviations for three separate determinations. c No activity detected. a

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TABLE 3. Kinetic parameters for the PC catalyzed formation of Pi in the presence of varying concentrations of pyruvatea.

kcat obs (s-1)

% WTobs

12.1 ± 0.4

(100)

T882S

0.146 ± 0.005

E218A

kcat calc interc

kcat calc intrad

Ka pyruvatee

kcat/Ka

intra

(mM)

(s-1 mM-1)

% WTcalc

inter

(s-1)

---

(100)

---

(100)

0.56 ± 0.07

21 ± 3

1.2

---

---

---

---

5.4 ± 0.6

0.027 ± 0.003

NAf

0

---

---

---

---

---

---

WT:T882S[1:1] HT

6.6 ± 0.1

55

6.1 ± 0.2

50.5

6.1 ± 0.2

50.5

0.91 ± 0.07

7.3 ± 0.6

WT:E218A[1:1] HTg

3.78 ± 0.07

31

3.1 ± 0.1

26

6.0(5) ± 0.2

50

NDh

ND

0.118 ± 0.003

0.9

0.133 ± 0.004

1.1

0.073 ± 0.002

0.6

0.37 ± 0.05

0.32 ± 0.04

WT

T882S:E218A[1:1] HT

(s-1)

% WTcalc

a

Reaction conditions: Tricine (100 mM, pH 7.5, 25 °C), HCO3- (40 mM), pyruvate (0-30 mM), MgATP (2.5 mM), MgCl2 (2.5 mM), acetyl-CoA (0.25 mM), MESG (0.2 mM), PNP (3-15 U). bInitial velocities at each concentration of pyruvate were determined in triplicate. Kinetic parameters are derived from fits of all data points to eqn 2 using non-linear regression. Errors reported are the standard deviation determined from the best-fit to eqn 2. cActivity calculated based on the probability of tetramer arrangements and relative activity of monomers within the tetramer for an intermolecular control mechanisms as described in the methods section. a Activity calculated based on the probability of tetramer arrangements and relative activity of monomers within the tetramer for an intramolecular control mechanisms as described in the methods section. eApparent Ka. f No activity detected. g Specific activities for the WT:E218A[1:1] HT were determined at 40 mM of pyruvate and saturating concentrations of all other substrates and activators. h Not determined.


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Biochemistry

TABLE 4. Ratios of kcat (s-1) and kcat/Km pyruvate (s-1 mM-1) for oxaloacetate formation (OAA) to pyruvate-stimulated Pi release (Pi).

(kcat)OAA/

(kcat/Km)OAA/

(kcat)Pi

(kcat/Ka)Pi

0.85 ± 0.03a

2.2 ± 0.4

1.1 ± 0.9

4.8 ± 0.9

WT:E218A[1:1] HT

0.85 ± 0.02

NDb

WT:T882S[1:1] HT

0.49 ± 0.01

1.4 ± 0.2

T882S:E218A[1:1] HT

0.64 ± 0.02

1.2 ± 0.2

WT T882S

a

Reported errors are propagated errors determined using the standard deviations for each kinetic parameter obtained from non-linear regression analysis. bNot Determined.


Biochemistry

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ASSOCIATED CONTENT. Supporting Information. Supporting information including probability analysis and additional kinetic data (Figs S1-S6) are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION. Corresponding Author *To whom correspondence should be addressed: Dr. Tonya N. Zeczycki, Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, East Carolina Heart Institute, 115 Heart Drive, Greenville, NC 27834, telephone: (252) 7445609; FAX (252) 744-3383; E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Funds provided by the Brody Brothers Endowment Grant and the Office of Graduate Studies and Research at East Carolina University. ACKNOWLEDGMENT. The authors would like to thank Dr. Mel Friske, professor of mathematics at Wisconsin Lutheran College, for his helpful discussion regarding probability theory. ABBREVIATIONS.


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Biochemistry

Biotin carboxylase, BC; Biotin carboxyl carrier protein, BCCP; carboxyl transferase, CT; HT, Hybrid tetramer; Pyruvate carboxylase, PC; Rhizobium etli pyruvate carboxylase, RePC. REFERENCES. [1] Jitrapakdee, S., and Wallace, J. (1999) Structure, function and regulation of pyruvate carboxylase, Biochem. J 340, 1-16. [2] Jitrapakdee, S., Vidal-Puig, A., and Wallace, J. C. (2006) Anaplerotic roles of pyruvate carboxylase in mammalian tissues, Cell. Mol. Life Sci. 63, 843-854. [3] Jitrapakdee, S., Wutthisathapornchai, A., Wallace, J. C., and MacDonald, M. J. (2010) Regulation of insulin secretion: role of mitochondrial signalling, Diabetologia 53, 10191032. [4] Jitrapakdee, S., St Maurice, M., Rayment, I., Cleland, W. W., Wallace, J. C., and Attwood, P. V. (2008) Structure, mechanism and regulation of pyruvate carboxylase, Biochem. J 413, 369-387. [5] St Maurice, M., Reinhardt, L., Surinya, K. H., Attwood, P. V., Wallace, J. C., Cleland, W. W., and Rayment, I. (2007) Domain architecture of pyruvate carboxylase, a biotindependent multifunctional enzyme, Science 317, 1076-1079. [6] Lietzan, A. D., Menefee, A. L., Zeczycki, T. N., Kumar, S., Attwood, P. V., Wallace, J. C., Cleland, W. W., and St. Maurice, M. (2011) Interaction between the biotin carboxyl carrier domain and the biotin carboxylase domain in pyruvate carboxylase from Rhizobium etli, Biochemistry 50, 9708-9723. [7] Zeczycki, T. N., Menefee, A. L., Jitrapakdee, S., Wallace, J. C., Attwood, P. V., St. Maurice, M., and Cleland, W. W. (2011) Activation and inhibition of pyruvate carboxylase from Rhizobium etli, Biochemistry 50, 9694-9707. [8] Yu, L. P. C., Xiang, S., Lasso, G., Gil, D., Valle, M., and Tong, L. (2009) A Symmetrical Tetramer for S. aureus Pyruvate Carboxylase in Complex with Coenzyme A, Structure 17, 823-832. [9] Lasso, G., Yu, L. P., Gil, D., Lázaro, M., Tong, L., and Valle, M. (2014) Functional Conformations for Pyruvate Carboxylase during Catalysis Explored by Cryoelectron Microscopy, Structure 22, 911-922. [10] Xiang, S., and Tong, L. (2008) Crystal structures of human and Staphylococcus aureus pyruvate carboxylase and molecular insights into the carboxyltransfer reaction, Nature Struct. Mol. Bio. 15, 295-302. [11] Zeczycki, T. N., St Maurice, M., Jitrapakdee, S., Wallace, J. C., Attwood, P. V., and Cleland, W. W. (2009) Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli, Biochemistry 48, 4305-4313. [12] Duangpan, S., Jitrapakdee, S., Adina-Zada, A., Byrne, L., Zeczycki, T. N., St. Maurice, M., Cleland, W. W., Wallace, J. C., and Attwood, P. V. (2010) Probing the catalytic roles of Arg548 and Gln552 in the carboxyl transferase domain of the Rhizobium etli pyruvate carboxylase by site-directed mutagenesis, Biochemistry 49, 3296-3304. [13] Zeczycki, T. N., Menefee, A. L., Adina-Zada, A., Jitrapakdee, S., Surinya, K. H., Wallace, J. C., Attwood, P. V., St. Maurice, M., and Cleland, W. W. (2011) Novel insights into the biotin carboxylase domain reactions of pyruvate carboxylase from Rhizobium etli, Biochemistry 50, 9724-9737.


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[14] Adina-Zada, A., Sereeruk, C., Jitrapakdee, S., Zeczycki, T. N., St. Maurice, M., Cleland, W. W., Wallace, J. C., and Attwood, P. V. (2012) Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase, Biochemistry 51, 8208-8217. [15] Menefee, A. L., and Zeczycki, T. N. (2014) Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase, FEBS J 281, 1333-1354. [16] Easterbrook-Smith, S. B., Hudson, P. J., Goss, N. H., Keech, D. B., and Wallace, J. C. (1976) Pyruvate carboxylase: Mechanism of the second partial reaction, Arch. Biochem. Biophys.176, 709-720. [17] Wallace, J. C., Phillips, N. B., Snoswell, M. A., Goodall, G. J., Attwood, P. V., and Keech, D. B. (1985) Pyruvate Carboxylase: Mechanisms of the Partial Reactionsa, Annals of the New York Academy of Sciences 447, 169-188. [18] Goodall, G. J., Baldwin, G. S., Wallace, J. C., and Keech, D. B. (1981) Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase, Biochem. J 199, 603-609. [19] WARREN, G. B., and TIPTON, K. F. (1974) The Role of Acetyl‐CoA in the Reaction Pathway of Pig‐Liver Pyruvate Carboxylase, European Journal of Biochemistry 47, 549554. [20] Warren, G. B., and Tipton, K. F. (1974) Pig liver pyruvate carboxylase. The reaction pathway for the decarboxylation of oxaloacetate, Biochem. J 139, 321-329. [21] Hill, T. L., and Levitzki, A. (1980) Subunit neighbor interactions in enzyme kinetics: halfof-the-sites reactivity in a dimer, PNAS 77, 5741-5745. [22] Lietzan, A. D., and St. Maurice, M. (2013) A Substrate-induced Biotin Binding Pocket in the Carboxyltransferase Domain of Pyruvate Carboxylase, J. Biol. Chem. 288, 1991519925. [23] Lietzan, A. D., and Maurice, M. S. (2013) Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs, Biochem. Biophys. Res. Comm. 441, 377-382. [24] Cheung, Y.-F., and Walsh, C. (1976) Studies on the intramolecular and intermolecular kinetic isotope effects in pyruvate carboxylase catalysis, Biochemistry 15, 3749-3754. [25] Khew-Goodall, Y. S., Johannssen, W., Attwood, P. V., Wallace, J. C., and Keech, D. B. (1991) Studies on dilution inactivation of sheep liver pyruvate carboxylase, Arch. Biochem. Biophys. 284, 98-105. [26] Upson, R. H., Haugland, R. P., Malekzadeh, M. N., and Haugland, R. P. (1996) A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate, Anal. Biochem. 243, 41-45. [27] Webb, M. R. (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems, PNAS 89, 4884-4887. [28] Adina-Zada, A., Jitrapakdee, S., Wallace, J. C., and Attwood, P. V. (2014) Coordinating Role of His216 in MgATP Binding and Cleavage in Pyruvate Carboxylase, Biochemistry 53, 1051-1058. [29] Marlier, J. F., Cleland, W. W., and Zeczycki, T. N. (2013) Oxamate is an alternative substrate for pyruvate carboxylase from Rhizobium etli, Biochemistry 52, 2888-2894.


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TOC GRAPHICS.


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Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate

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