Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of

Subscriber access provided by EAST TENNESSEE STATE UNIV

Article

Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase Lauren E Westerhold, Lance C Bridges, Saame Raza Shaikh, and Tonya Nicole Zeczycki Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00383 • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 17, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Kinetic and Thermodynamic Analysis of AcetylCoA Activation of Staphylococcus aureus Pyruvate Carboxylase Lauren E. Westerhold1,2, Lance C. Bridges3, Saame Raza Shaikh1,2 and Tonya N. Zeczycki1,2*

1

Department of Biochemistry and Molecular Biology and the 2East Carolina Diabetes and

Obesity Institute, Brody School of Medicine at East Carolina University, Greenville, NC, 27834. 3

Department of Biochemistry, Molecular and Cell Sciences, Arkansas College of Osteopathic

Medicine,

Arkansas

Colleges

of

Health

Education,

Ft.

Smith,

AR,

72916.

ACS Paragon Plus Environment

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 50

ABSTRACT. Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC) catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linkedfunction, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetylCoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism

by

which

acetyl-CoA

coordinates

catalysis

within

the

PC

tetramer.


2

Page 3 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

INTRODUCTION. Allosteric control of enzyme activity is a critical aspect of enzyme function; however, dysregulated enzyme activity is often a contributing factor to the pathogenesis of numerous diseases. For example, uncontrolled pyruvate carboxylase (PC) activity correlates with increases in hepatic gluconeogenesis in type 2 diabetics

1-3

and growth

and invasion in metastatic breast cancer 4. Properly regulated enzyme activity can also contribute to disease pathogenesis. For example, regulated PC activity in Listeria monocytogenes

5-7

and

Staphylococcus aureus 8 is directly linked with the organism’s pathogenicity. In fact, a genomewide analysis examining the biosynthetic pathways required for full Staphylococcus virulence showed that mutations in the pycA gene (encoding for PC) resulted in significant attenuation of Staphylococcus infection 8. This decisive role of PC in the pathogenesis of metabolic diseases and the pathogenicity of Staphylococcus infection underscores the need to delineate the allosteric mechanisms governing PC activity to develop effective therapeutics that modulate PC activity. At the molecular level, however, the allosteric mechanisms and associated conformational changes governing PC activity are not well defined. In the α4 PC family, which includes PC from Staphylococcus aureus, Rhizobium elti (RePC) and humans, a single, ~125-130 kDa MW polypeptide chain contains all four functional domains, namely the biotin carboxylase (BC), carboxyl transferase (CT), biotin carboxyl carrier protein (BCCP) and allosteric 9, or tetramerization 10, domains. Acetyl-CoA, which binds in the allosteric domain, is the most potent activator of the α4 PCs isolated from a variety of sources, including mammals, yeast and bacteria 11, 12

and is often essential for full activity. The dimer of dimers arrangement of monomers in the

tetramer creates two catalytically distinct faces where two monomers are antiparallel relative to each other on a single face of the tetramer and perpendicular to the additional monomers on the


3

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

opposing face (Figure 1)

9, 13

Page 4 of 50

. The global architecture of the tetramer is such that two of the

possible four acetyl-CoA binding sites are contained within a single face of the tetramer.

Figure 1. The overall architecture of the Staphylococcus aureus (Sa) PC tetrameric holoenzyme (pdb ID 3BG514). A single monomer is shown in cartoon representation. 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, not shown due to high disorder). Biotin is covalently attached to a strictly conserved Lys residue in the BCCP domain. Acetyl-CoA is shown as sticks bound in the allosteric domain. Monomers on a single face (monomer 1 and 2) are positioned antiparallel relative to each other and perpendicular to monomers 3 and 4 on the opposing face. Catalysis occurs between BC and CT domains on opposing monomers on the same face (i.e. between monomers 1 and 2, black dashed arrows) Crystal structure figures were generated using PyMOL Molecular Graphics System (v1.8, Schrödinger, LLC).


4

Page 5 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

High-resolution crystal structures

9, 10, 14

and cryo-EM studies

15, 16

have highlighted the

relative positioning of the activator in the allosteric domain (Figure 1) and the constellation of residues important to promoting the allosteric response in PC and facilitating catalytic turnover 17-19

. The kinetic mechanisms associated with activity in the BC and CT domains are well-

defined

20-25

. Briefly, catalysis occurs through an intermolecular mechanism with the mobile

BCCP domain translocating from the BC domain on its own monomer to the CT domain on the opposing monomer 9, 26, 27. A biotin cofactor, covalently tethered to the BCCP domain, is initially carboxylated in the BC domain to form a carboxybiotin intermediate. The intermolecular translocation of the BCCP domain shuttles the intermediate to a neighboring CT domain ~65 Å away 14, 15, 27, 28. Subsequent carboxybiotin decarboxylation in the CT domain active site liberates CO2, which, upon nucleophilic attack by the purported pyruvate-enolate intermediate, generates oxaloacetate (Scheme 1). Scheme 1. Reactions scheme for PC-catalyzed carboxylation of pyruvate. Biotin carboxylation (1) occurs in the BC domain and pyruvate carboxylation (2) occurs in the CT domain.

Pre-steady

29

and steady-state kinetics

18, 21, 25

show that the presence of acetyl-CoA

increases the amount of productive MgATP-cleavage in the BC domain, most likely by promoting the proper positioning of MgATP and residues within the active site and facilitating


5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 50

the movement of the mobile-carboxyl carrier domain 21, 28, 30. While these previous studies have revealed the location and relative impact acetyl-CoA has on the activity in each of the domains, we still have a poor understanding of how the potent activator acetyl-CoA synchronizes catalytic events occurring in the spatially discrete BC and CT domains. Recent mutagenic studies of PC isolated from Rhizobium etli

17, 31

suggest that acetyl-

CoA aids in constraining and stabilizing global symmetric-to-asymmetric conformational changes in the PC tetramer that are assumed to be associated with catalytic turnover in a “halfthe-sites” active catalytic model. Cryo-EM images of SaPC

15, 16

suggest that PC adopts an

asymmetric conformation when the BCCP domain is associated the BC domain (i.e. biotin carboxylation) and a symmetrical conformation when the BCCP domain is associated with the CT domain (i.e. pyruvate carboxylation) 15. Previous mutagenic studies suggest that acetyl-CoA enhances the rate of turnover by stabilizing one of the conformers through interactions with both conserved and non-conserved residue networks

17, 31

. In line with this hypothesis, we reason that the intrafacial interactions

between the allosteric domains of monomers on opposing polypeptide chains establish a direct communication network between the CT and BC domains. We hypothesize that acetyl-CoA plays an additional role in coordinating intersubunit communication

32

by energetically and

kinetically coupling the substrates of the CT and BC domains, presumably through the communication networks established between the allosteric and BC/CT domains. Herein, we report a kinetic and thermodynamic analysis of the impact of acetyl-CoA on the activation of the SaPC catalyzed carboxylation of pyruvate. Our results show that acetyl-CoA and MgATP bind to SaPC in a random fashion prior to the addition of pyruvate. We additionally show that all three ligands are energetically coupled. Interestingly, the absence of acetyl-CoA


6

Page 7 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

completely abolishes the thermodynamic linkage between MgATP and pyruvate, suggesting a functional role in facilitating long-range communication by energetically coupling substrates in the BC and CT domains. The thermodynamic activation parameters were largely independent of acetyl-CoA, with the exception of the activation entropy (∆S‡). Our thermodynamic results also lend further support to the idea that the symmetric and asymmetric PC conformations are differentially stabilized by species-specific residue interactions at the dimer-dimer interface 10, 14 and interactions between acetyl-CoA and non-conserved residues in the allosteric domain

31

.

Taken as a whole, our results further define the functional role for acetyl-CoA in coordinating conformational dynamics with catalytic activity. METHODS Materials. IPTG, biotin, NADH, kanamycin and chloramphenicol were purchased from Research Products International Corp. (RPI). Ni2+-Profinity IMAC resin was obtained from BioRad. Pyruvate (sodium salt) was obtained from Fisher Scientific and the trilithium salt of acetylCoA 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. SaPC protein expression and purification. Recombinant PC from Staphylococcus aureus (SaPC) was co-overexpressed in Escherichia coli BL21 Star (DE3) with BirA for biotinylation as previously described 14, 15. For protein purification, cell paste was thawed at 4 °C with continuous stirring in lysis buffer containing 20 mM HEPES, 10 mM imidazole, 10 mM MgCl2, 200 mM NaCl, 1 mM TCEP, 200 µg/mL lysozyme, and 1 mM PMSF. Cells were lysed on ice via sonication (Branson Digital Sonifier, 6 min, max temp 10 °C, 80% amplitude, pulse 59.9 s, pulse off 30 s). All purification steps were performed at 4 °C. The lysate was clarified via


7

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 50

centrifugation (12,000 x g for 15 min) and loaded (1.5 ml/min) onto 25 mL of Profinity IMAC Ni2+- Charged Resin (Bio-Rad) that was equilibrated with 5 column volumes (CV) of wash buffer containing 20 mM HEPES (pH 8.0), 10 mM MgCl2, 20 mM imidazole, 200 mM NaCl, and 1 mM TCEP. The protein was eluted using a 100 mL linear imidazole gradient (20 mM – 300 mM). Fractions containing SaPC were identified using SDS-PAGE, pooled and dialyzed overnight against 2 L of dialysis buffer (2 x 1 L, 10 mM HEPES, 10 mM MgCl2, 50 mM NaCl, and 1 mM TCEP). The dialyzed protein was then loaded (1.5 mL/min) onto 25 mL of QSepharose Fast Flow Resin (GE Healthcare) that was equilibrated with 5 CV of wash buffer containing 20 mM HEPES, 10 mM MgCl2, 50 mM NaCl, and 1 mM TCEP. The protein was eluted using a 100 mL linear NaCl gradient (50 mM – 1 M). Fractions containing PC were identified using SDS-PAGE and dialyzed overnight against 2 L of dialysis buffer (2 x 1 L, 10 mM HEPES, 10 mM MgCl2, 50 mM NaCl, 1 mM TCEP, and 0.1 mM NaN3). The purified enzyme was then concentrated to ~ 3 mg/mL using a pressure-based concentration cell (EMD Millipore), flash-frozen in 250 µL aliquots and stored at -80 °C until used. Total protein concentration was determined using a BCA Protein Assay Kit (Pierce). Complete biotinylation (> 99%) of the SaPC protein was confirmed using an avidin-based gel shift assay 28. Enzyme activity assays. The initial rates of oxaloacetate formation from the MgATPdepdendent carboxylation of pyruvate by HCO3- were determined spectrophotometrically using a Shimadzu UV-1800 Spectrophotometer equipped with a 6 Cell-Thermoelectrical Temperature Controller. All reactions were performed in 1 mL reaction volumes at pH 7.6 (100 mM Tricine) and 25 °C unless otherwise noted. Rates of pyruvate carboxylation to form oxaloacetate 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)28. All stock


8

Page 9 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

solutions were made fresh prior to use and the stock concentrations of substrates and activators (i.e.

MgATP,

pyruvate

and

acetyl-CoA)

were

determined

using

end-point

and

spectrophotometric assays as previously described21, 28, 32. Determination of kcat and apparent kcat/Km for MgATP, pyruvate and acetyl-CoA for the SaPC catalyzed formation of oxaloacetate. To determine the kcat (sec-1) and kcat/Km (sec-1 mM-1) associated with the SaPC catalyzed carboxylation of pyruvate for MgATP, pyruvate and acetylCoA, the initial rates of oxaloacetate formation were determined at varying concentrations of substrate or activator (MgATP, 0.025-2.5 mM; pyruvate, 0.025-40 mM; acetyl-CoA, 0-500 µM). Substrate or activator concentrations were varied one at a time while all other ligands were held at fixed and saturating concentrations (MgATP, 2.5 mM; pyruvate 15 mM; acetyl-CoA, 250 µM). Reactions were performed in 1 mL total volumes at 25 °C and contained 100 mM Tricine (pH 7.6), MgCl2 (2.5 mM), HCO3- (40 mM), NADH (0.2 mM), malate dehydrogenase (5 U). Reactions were initiated with the addition of ~3-150 µg of SaPC. Initial rates at each substrate or activator concentration were determined in triplicate and error bars are ± std dev. Initial velocity studies and thermodynamic linkage analysis. Initial velocities were measured at varying concentrations of one substrate at fixed concentrations of a second substrate or activator while the third substrate/activator was held constant at saturating concentrations. For example, the rates of oxaloacetate formation were measured across variable concentrations of pyruvate (0.25 – 15 mM) at fixed MgATP (0.05 – 2.5 mM) while acetyl-CoA was held constant at 250 µM. Acetyl-CoA concentrations, when varied, were between 0-250 µM. The 1 mL reactions also contained: 100 mM HCO3-, 5 mM MgCl2, 0.25mM NADH, and malate dehydrogenase (5 units). Reactions were initiated with the addition of ~0.01 – 1.36 mg of SaPC


9

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 50

and contained 100 mM Tricine (pH 7.6), pyruvate (15 mM), MgATP (2.5 mM), MgCl2 (2.5 mM), HCO3- (100 mM), NADH (0.2 mM), malate dehydrogenase (5 U). Linkage-analysis and determination of the free energies of interaction. Like the initial velocity studies, the rates of pyruvate carboxylation were measured over a wider range of varied ligand concentrations to determine the linkage equilibrium, or thermodynamic coupling, between MgATP, pyruvate and acetyl-CoA33-36. Individual data fits of the initial velocity plots to the Michaelis-Menten equation allowed for the determination of the apparent Km

pyruvate

values as a

function of increasing MgATP concentrations in the presence and absence of acetyl-CoA. Similarly, we determined the apparent Km for pyruvate or MgATP as a function of increasing acetyl-CoA concentrations. Determination of thermodynamic activation parameters (Ea, ∆G‡, ∆H‡ and ∆S‡) for pyruvate carboxylation in the absence and presence of saturating acetyl-CoA. To determine the thermodynamic activation parameters associated with pyruvate carboxylation, the specific activities for carboxylation at saturating substrate and activator concentrations were determined over a range of temperatures (15-55 °C). Reactions contained 100 mM Tricine (pH 7.6), pyruvate (15 mM), MgATP (2.5 mM), MgCl2 (2.5 mM), HCO3- (100 mM), NADH (0.2 mM), malate dehydrogenase (5 U), SaPC (10-75 µg). Initial velocities (vi, sec-1) was monitored as above using the malate dehydrogenase coupled assay system in the presence or absence of acetyl-CoA (250 µM). Specific activities were determined in triplicate at each temperature and error bars are ± std dev. Data Analysis. Determination of kcat and kcat/Km. Kinetic parameters (kcat and kcat/Km

pyruvate

or kcat/Km MgATP) for pyruvate carboxylation were determined by fitting initial velocities (vi) to


10

Page 11 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

the Michaelis-Menten equation (eqn 1) using nonlinear regression (Prism 7.0, GraphPad, San Diego CA)

vi =

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

where kcat (sec-1) is the rate of carboxylation at saturating substrate (A) 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 when acetyl-CoA was the varied ligand (kcat and kcat/K0.5 acetyl-CoA) were determined by fitting initial velocities to either eqn 2 or eqn 317 using nonlinear regression

vi =

k cat [acetyl-CoA]n K 0.5 +[acetyl-CoA]n (2)

vi = k 0 cat +

k cat   Ka 1+    [acetyl − CoA ] 

n

(3) where kcat (sec-1) is the rate of carboxylation at saturating acetyl-CoA, k0 cat (sec-1) is the rate of acetyl-CoA independent activity, Ka is the apparent affinity constant for the activator and n is the Hill coefficient. A comparison of data fits of the initial rates of pyruvate carboxylation as a function of [acetyl-CoA] to eqns 1-3 indicated that the Hill equation is the preferred model based


11

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

on the extra sum-of-squares F-test (Prism 7.0; p < 0.001)

Page 12 of 50

37

. Errors for the kinetic parameters

were derived from the fit to eqn 2 and are reported as ± std dev. Initial velocity studies. Initial velocity data were first analyzed graphically using double reciprocal plots of vi vs. concentration and the respective secondary plots

38, 39

. Data were then

globally fitted to the appropriate rate equation using non-linear regression. Data obtained for the systematic analysis of pyruvate carboxylation as a function of varying pyruvate and MgATP in the presence of saturating acetyl-CoA were best described and globally fitted to the equation describing a sequential mechanism (eqn 4) 39

vi =

k cat [A][B] K ia K B + K b [A] + K a [B] + [A][B] (4)

where kcat (sec-1) is the theoretical maximal rate of pyruvate carboxylation, A is the concentration of the varying substrate, B is the concentration of the fixed substrate, Kia is the apparent dissociation constant for A (i.e. low [B]), and Ka and Kb are the Michaelis-Menten constants for substrates A and B, respectively. Models were initially chosen based on graphical analysis of the initial velocity plots and secondary plots, as well as comparison of data fits to equations for an equilibrium ordered or ping-pong39 type mechanism. Global fits of the data to each equation were compared using extra sum-of-squares F-test (p < 0.001)37. In cases where the extra sum-ofsquares F-test results in similar Dfn values, Akaike’s Information Criteria (AIC) was used to further establish which model was most likely (< 99.98% probability) to generate the observed data40. Results from the global fits (goodness-of-fit statistics, confidence intervals (95%)) and statistical comparison of the models are provided in the supporting information (SI Tables S2S5). Kinetic parameters are reported as the mean ± std dev determined from global fits of the


12

Page 13 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

data to the preferred model. Kinetic parameters are reported as the mean ± std dev determined from non-linear regression. Plots of vi vs. [pyruvate] or [MgATP] and fixed [acetyl-CoA] were globally fitted to equations describing three different kinetic mechanisms for an enzyme with multiple activator sites (n), namely one where the activator binds prior to the substrate (eqn 5), the substrate binds prior to the activator (eqn 6) or the substrate and activator bind to the enzyme in a random manner (eqn 7) 41.  [S][ A ]n  k cat    K s K An    vi = n n  S][ A ]   [A]  [  1 +  + K A   K s K nA  

(5)

 [S][ A ]n  k cat    K s K nA    vi = n S]  [ A ]  [ 1+ 1+ K s  K A  (6)

 [S][ A ]n  k cat    K s K nA    vi = n S]   [ A ]   [ 1 +  1+ KS   K A   (7)

In all three equations, kcat (sec-1) is the theoretical maximal rate of pyruvate carboxylation, [S] is the concentration of the varied substrate (i.e. MgATP or pyruvate), [A] is the concentration of


13

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 50

the activator (i.e. acetyl-CoA), n is the theoretical number of activator molecules bound to the enzyme, similar to the Hill coefficient, Ks is the apparent Michaelis constant for the substrate and KA is the apparent Michaelis constant for the activator. Global fits of the data to each equation were compared using extra sum-of-squares F-test (p < 0.001)37. In cases where the extra sum-ofsquares F-test results in similar Dfn values, Akaike’s Information Criteria (AIC) was used to further establish which model was most likely (< 99.98% probability) to generate the observed data40. Results from the global fits (goodness-of-fit statistics, confidence intervals (95%)) and statistical comparison of the models are provided in the supporting information (SI Tables S2S5). Kinetic parameters are reported as the mean ± std dev determined from non-linear regression. Linked-function analysis and coupling free energies. To quantify the thermodynamic influence of one ligand (i.e. MgATP, pyruvate and acetyl-CoA) on the binding of another ligand, it was necessary for us to determine the coupling parameter (Q) between two ligands

33, 34, 36, 42

.

In this analysis, we have designated MgATP, pyruvate and acetyl-CoA as A, B and X, respectively. The coupling parameter (Q) between MgATP and acetyl-CoA in the presence of saturating pyruvate, designated Qax/b, is defined in eqn 843, 44 Qax/b =

K ia/b K = ix/b K ia/xb K ix /ab

(8) where Kia/b is the dissociation constant of substrate A (i.e. MgATP) in the absence of ligand X (i.e. acetyl-CoA) and the presence of saturating B (i.e. pyruvate). In this notation35, 36, the ligand designation following the slash is saturating, thus Kia/xb represents the dissociation constant of MgATP (A) in the presence of saturating acetyl-CoA (X) and pyruvate (B). Our steady-state


14

Page 15 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

kinetic analysis verified that rapid equilibrium assumption in the steady-state is valid for the carboxylation reaction34. The coupling parameters of Qax/b (MgATP and acetyl-CoA, saturating pyruvate) and Qbx/a (pyruvate and acetyl-CoA, saturating MgATP) were graphically estimated from plots of the apparent Km for each substrate as a function increasing acetyl-CoA45,

46

. To determine the

coupling parameters between MgATP and pyruvate in the presence of acetyl-CoA, Qab/x, plots of the apparent Km pyruvate vs. [MgATP] were fitted to eqn 9

 K ia/x + [ MgATP ]  K m pyruvate = K ib/x    K ia /x + Qab/x [ MgATP] 

(9) where Km pyruvate is the apparent Km determined at fixed concentrations of MgATP in the presence of saturating concentrations of acetyl-CoA, Kib/x is the apparent dissociation constant for pyruvate (B) in the absence of MgATP (A) and saturating acetyl-CoA (X) and Kia/x is the apparent dissociation constant for MgATP in the absence of pyruvate. In the absence of acetylCoA, there was no observed dependence of the apparent Km pyruvate on MgATP concentration (Qab = 1). The coupling constants describing the cooperative interactions between MgATP, pyruvate and acetyl-CoA are equilibrium constants43, 47 for the disproportion reactions shown in

Scheme 2. Scheme 2. Disproportion reactions for the cooperative interactions (Q) between SaPC (PC) MgATP, pyruvate (pyr) and acetyl-CoA (ACoA).


15

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 50

As such, the free energies of interaction43, 44 between MgATP and acetyl-CoA (∆Gax/b) , pyruvate and acetyl-CoA (∆Gbx/a) and MgATP and pyruvate in the presence of saturating acetyl-CoA (∆Gab/x) are defined by the following relationships (eqns 10-12)

G ax/b = − RT ln ( Qax /b ) (10)

G bx/a = − RT ln ( Qbx / a ) (11)

G ab/x = − RT ln ( Qab/ x ) (12) where ∆G is the coupling free energies between two ligands in the presence of saturating concentrations of the third, R is the universal gas constant in J mol-1 K-1, T is the temperature in K (298 K) and Q is the coupling parameters determined in the linkage analysis.


16

Page 17 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Thermodynamic activation parameters. To determine the Ea for the carboxylation reaction, Arrhenius plots of 1/T (K) vs. –R (ln vi) were constructed. Data were fitted to the linear form of the Arrhenius equation (eqn 13) 1 − R ( ln k obs ) = E a   + ln A T

(13) where R is the universal gas constant in J mol-1 K-1, kobs (sec-1) is the specific activities observed at saturating concentrations of substrate in the presence or absence of saturating acetyl-CoA, Ea is the activation energy for the enzymatic reaction, T is temperature in °K and A is the preexponential factor. Similarly, Eyring plots of ln (kobs /T) vs 1/T were constructed to determine the enthalpy (∆H‡) and entropy (∆S‡) of activation directly. Data were fitted to eqn (14) ‡ ‡ k  H 1  k′   S  ln  obs  = + ln +        T  R T h R

(14) where kobs (sec

-1

) is the specific activities for pyruvate carboxylation determined at each

temperature (T, °K) in the presence or absence of saturating acetyl-CoA, R is the universal molar gas constant in J mol-1 K-1, ∆H‡ is the enthalpy of activation (kJ mol-1), k’ is the Boltzmann constant, h is Planck’s constant and ∆S‡ is the entropy of activation (J mol-1 K). Thermodynamic parameters from both the Arrhenius and Eyring plots are reported as mean ± std dev determined from the non-linear regression.

RESULTS. Determination of apparent kinetic parameters (kcat and kcat/Km) for pyruvate, MgATP and acetyl-CoA. To determine preliminary estimates for the apparent kcat (sec-1) and kcat/Km (sec-1


17

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 50

mM-1) for pyruvate, MgATP and acetyl-CoA in the forward reaction, we monitored the initial rates (vi) of pyruvate carboxylation as a function of varying substrate or activator concentration at constant, saturating levels of all other substrates and activators using the malate dehydrogenase coupled assay system. The vi vs. [concentration] plots when pyruvate or MgATP were varied are hyperbolic and the double-reciprocal plots were linear (Figure S1), indicating Michaelis-Menten type kinetics (Table 1).

Table 1. Kinetic parameters determined for the MgATP-dependent carboxylation of pyruvate by HCO3- catalyzed by SaPCa. kcat

kcat/Km or kcat/K0.5

Km or K0.5 b

(sec-1)

(sec-1 mM-1)

(mM)

Pyruvate

2.5 ± 0.5

5.01 ± 0.07

0.50 ± 0.04

MgATP

3.0 ± 0.1

8.3 ± 0.1

0.36 ± 0.05

2.58 ± 0.05

266.67 ± 0.06

0.0097 ± 0.0006

Acetyl-CoAc

a

Reaction conditions: 100 mM Tricine (pH 7.4, 25 °C) pyruvate (0.25-15 mM), MgATP (0.052.5 mM), acetyl-CoA (0.001-0.250 mM), MgCl2 (2.5 mM), HCO3- (40 mM), NADH (0.2 mM), malate dehydrogenase (5 U), SaPC (10-50 µg). The kcat and apparent kcat/Km were determined by measuring the initial rates of carboxylation at varied concentrations of a single substrate or activator. All other substrates/activators were held at constant, saturating concentrations (MgATP, 2.5 mM; pyruvate, 15 mM, acetyl-CoA, 0.25 mM). Initial rates were determined in triplicate and reported error is ± std dev for each parameter determined from fits to eqn (1) using nonlinear regression. bApparent Km. cData were fitted to eqn (2) using nonlinear regression as above with a hill value (n) of 1.4 ± 0.2. Reported error is ± std dev for each parameter determined from nonlinear regression. Since the apparent kcat obtained herein at saturating levels of acetyl-CoA was lower than that previously reported for the SaPC catalyzed formation of oxaloacetate

10

, we determined the

specific activities of carboxylation under conditions similar to those used in the previous study, notably in the presence of Tris (100 mM) and NaCl (200 mM). Both Tris and Na+ are known


18

Page 19 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

activators of PC

48

(15.7 ± 0.6 sec-1

. Specific activities (14.23 ± 0.07 sec -1) were comparable to reported values

10

), suggesting that the difference in rates are due to the inclusion of the

activators in previous studies. In contrast to the hyperbolic dependence of the rate on pyruvate and MgATP concentrations, we observed a sigmoidal dependence of vi on acetyl-CoA concentration. A comparison of fits, using the extra-sum-of-squares F-test 37, of the vi for pyruvate carboxylation as a function of [acetyl-CoA] to the Michaelis-Menten equation (eqn 1), the Hill equation (eqn 2) and a modified Hill equation accounting for acetyl-CoA independent activity (eqn 3 17) indicated that the data is best described by the Hill equation (p < 0.001). The K0.5 for acetyl-CoA was 9.7 ± 0.6 µM with a Hill coefficient (n) of 1.4 ± 0.2. Initial velocity studies and pairwise analysis in the forward reaction. Pyruvate vs. MgATP, saturating acetyl-CoA. To define the kinetic mechanism, initial velocity patterns were obtained by measuring the rate of oxaloacetate production as a function of varying concentrations of one ligand (i.e. MgATP, pyruvate and acetyl-CoA) at fixed concentrations of the second and saturating concentrations of the third. Plots of vi vs [pyruvate] at fixed MgATP (Figure 2A) or vi vs. [MgATP] at fixed pyruvate (Figure 2B) and saturating acetyl-CoA were hyperbolic and double reciprocal plots (Figure 2C and D) were linear. Similar to previous studies 49, 50, we observed a slight discontinuity in the secondary plots (Figures S2-3) most likely due to non-productive MgATP cleavage and incomplete coupling between the BC and CT domain reactions at low pyruvate concentrations

28, 49, 50

. The intersecting pattern of the double

reciprocal plots and linearity of the secondary plots suggest the sequential, ordered addition of ligands in the presence of acetyl-CoA 51.


19

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 50

Figure 2. Initial velocity plots for the SaPC catalyzed carboxylation of pyruvate determined in the presence of saturating acetyl-CoA. (A) Rate of pyruvate carboxylation (vi, sec-1) as a function of varying [pyruvate] (0.25-15 mM) at fixed [MgATP] (●, 0.05 mM; ■, 0.1 mM; ▲, 0.25 mM;▼, 0.375 mM; ♦, 0.5 mM; ●, 0.75 mM; ■, 1 mM; ▲, 1.5 mM;▼, 2 mM; and ♦, 2.5 mM). Data were individually fitted to the Michaelis-Menten equation (eqn 1) and dashed lines are the best-fit lines determined via non-linear regression. (B) Double reciprocal plots for (A). Symbols and colors are the same as in (A). Data were individually fitted to a straight line (dashed lines) for secondary plot analysis (Figure S2). (C) Rate of pyruvate carboxylation (vi, sec-1) as a function of varying [MgATP] (0.05-2.5 mM) at fixed [pyruvate] (●, 0.25 mM; ■, 0.5 mM; ▲, 1 mM;▼, 1.5 mM; ●, 3 mM; ■, 5 mM; ♦, 7 mM; and ▲, 15 mM). Data were individually fitted to the Michaelis-Menten equation (eqn 1) and dashed lines are the best-fit lines determined via non-linear regression. (D) Double reciprocal plots. Symbols and colors are the same as in (C).


20

Page 21 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Data were individually fitted to a straight line (dashed lines) for secondary plot analysis (Figure S2).

As such, data were globally fitted to a sequential model (Figure 3, Table 2) and the resulting kinetic parameters were consistent when either [pyruvate] or [MgATP] were varied. The apparent Km for pyruvate (0.37 ± 0.05 mM) and MgATP (0.19 ± 0.02 mM) are comparable with those determined previously for SaPC 10, 14 and indicate that, in the presence of saturating acetylCoA, MgATP adds to SaPC in the BC domain active site prior to pyruvate addition in the CT domain.

Figure 3. Global fits of the initial velocity plots for the SaPC catalyzed carboxylation of pyruvate determined in the presence of saturating acetyl-CoA. (A) Rate of pyruvate carboxylation (vi, sec-1) as a function of varying [pyruvate] (0.25-15 mM) at fixed [MgATP] (●, 0.05 mM; ■, 0.1 mM; ▲, 0.25 mM;▼, 0.375 mM; ♦, 0.5 mM; ●, 0.75 mM; ■, 1 mM; ▲, 1.5 mM;▼, 2 mM; and ♦, 2.5 mM). Data were globally fitted to eqn 4 (p 2.5 mM resulted in severe substrate

inhibition 19 in the absence of acetyl-CoA (data not shown) and prevented examining the reaction at concentrations > 2.5 mM. Thermodynamic linkage analysis. To quantitatively asses the allosteric and cooperative influence of acetyl-CoA, we used thermodynamic linkage analysis to determine the coupling parameters (Q) and free energies of interaction

33-36, 42

, between MgATP, pyruvate and acetyl-

CoA. Based on the principles of linked equilibria and reciprocity

58-60

, the phenomena of

thermodynamic linkage between two ligands (X and Y) binding at discrete sites on the protein indicates that if the affinity of the protein for ligand X is altered when ligand Y is bound, then the affinity of the protein for Y must be altered by the same amount when X is bound. The binding


28

Page 29 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

interactions of the two ligands are therefore “linked” by the magnitude (Q) of this proteinmediated interaction. Accordingly, the free energies and coupling parameters determined are related to the conformational changes or conformational equilibria induced by ligand binding 35, 59

. An analysis of the linked equilibrium responsible for the allosteric or cooperative

interactions between the ligands in the SaPC catalyzed carboxylation of pyruvate considers all the enzyme complexes in a thermodynamic cycle

33, 35

. For example, Scheme 4 shows the

enzyme species considered in the linkage analysis between MgATP (A) and acetyl-CoA (X) in the presence of saturating pyruvate (B). Because of the saturating concentrations of pyruvate, the four-enzyme species considered are the SaPC-pyruvate (EB), SaPC-pyruvate-acetyl-CoA (EBX), SaPC-pyruvate-MgATP (EBA) and activated ternary complex (EBXA).

Scheme 4. Representative thermodynamic cycle for the linked equilibrium analysis of coupling between MgATP (A) and acetyl-CoA (X) in the presence of saturating pyruvate (B).

The four dissociation constants shown in Scheme 4 are not independent, since the free energy (∆Gaxb) associated with the conversion of EB to EBAX is the same regardless of whether


29

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MgATP or acetyl-CoA binds to SaPC first

Page 30 of 50

58-60

. From a structural standpoint, this principle

implies that the conformational changes associated with the interconversion of the enzyme species are also interdependent, meaning that the free energies of interaction associated with the ligand induced conformational changes are independent of the pathway taken, but dependent on which ligands are present in the respective binding sites. Using the example provided in Scheme

4, this means that conformational changes induced by the binding of MgATP to SaPC at saturating concentrations of acetyl-CoA are the same whether acetyl-CoA or MgATP bind first. The difference in the coupling energies, which is equal to the ratio of the dissociation constants in the presence or absence of the coupled ligand (eqn 8) is defined as Q and quantifies the allosteric or cooperative effect 35. If Q is >1, the coupling results in activation and if Q = 1, there is no thermodynamic coupling between the two ligands, and ultimately no allosteric effect since Kia = Kia/x. Similar thermodynamic cycles exist for each of the scenarios examined (i.e. the coupling between MgATP and pyruvate in the presence and absence of acetyl-CoA and coupling between pyruvate and acetyl-CoA in the presence of saturating MgATP) and Scheme 2 shows the designated coupling parameter (Q) for each of the unique disproportion reactions. Based on the apparent Michaelis constants and coupling parameters determined below, we can assume that the substrates effectively achieve a binding equilibrium with SaPC in the steady-state34. In addition, we note that there is the possibility of numerous inter- and intramolecular coupling interactions within the SaPC tetramer, including homotropic couplings between ligands contained within a single dimer face or between the two faces of the tetramer. To simplify the analysis, we used a single modifier, single activator symmetrical dimer model 42 and assumed that homotropic interactions between the ligands were negligible. While these assumptions mean we cannot isolate specific energetic couplings (i.e. between acetyl-CoA on


30

Page 31 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

one monomer with MgATP on the same monomer in a single face), we can, however, generate a global picture of the thermodynamic equilibrium couplings underpinning the allosteric response of SaPC. We observed a positive Q value for the coupling interactions between MgATP or pyruvate and acetyl-CoA in the presence of saturating concentrations of the other ligand, i.e. Qax/b and Qbx/a, respectively, indicating allosteric activation (Figure 6). The data was not well described by eqn 9, most likely because of the cooperative acetyl-CoA binding to SaPC observed in the sigmoidal rate vs. [acetyl-CoA] curves (Figures S1 and 4) 46. Therefore, we estimated the dissociation constants from the graphical analysis of apparent Km vs. [acetyl-CoA] curves, similar to methods previously employed for phosphofructokinase

46, 47, 61

, and then used the

relationship in eqn 8 to determine a reasonable estimate for Q (Figure 6, Table 4). The magnitude of the coupling between the ligands and acetyl-CoA was similar (Qax/b = 5.8 ± 0.1 and Qbx/a = 6.9 ± 0.4).


31

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 50

Figure 6. Variation of the apparent Km for MgATP and pyruvate as a function of acetyl-CoA concentration. (A) Plots of apparent Km for MgATP (●) and pyruvate (●) as a function of varied acetyl-CoA. (B) Log-Log plots of apparent Km vs [acetyl-CoA]. The cooperativity of the acetylCoA binding prevented these data from being fit to eqn 9, but estimates of Qax/b and Qbx/a were made from the limiting values (dashed lines) of Kia/b and Kiax/b and Kib/a and Kibx/a, respectively46. The magnitude of Qbx/a and Qax/b are represented by the vertical arrows. Values for the coupling parameters are show in Table 4.


32

Page 33 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Table 4. MgATP, pyruvate and acetyl-CoA coupling constants (Q) and free energies of binding (∆G) that quantify the interactions between the three ligands on SaPC.a

Interacting Ligands

Other Saturating Ligand

Designation Q

∆G at 25 °C

(kJ mol-1)

MgATP – Acetyl- Pyruvate CoA

Qax/b

5.8 ± 0.1 -4.35 ± 0.08

Pyruvate – Acetyl- MgATP CoA

Qbx/a

6.9 ± 0.4 -4.8 ± 0.1

MgATP – Pyruvate

Acetyl-CoA

Qab/x

5.5 ± 0.2 -4.23 ± 0.09

MgATP – Pyruvate

---

Qab

1

1

a

Initial rates were determined in triplicate and reported error are ± std dev determined as described in the data analysis section.

The coupling between MgATP and pyruvate in the presence and absence of acetyl-CoA in a similar manner and the data were fitted to eqn 9 to obtain Qab/x (Qab/x = 5.5 ± 0.2). Again, we observed a similar magnitude of interaction between the two substrates in the presence of acetylCoA (Figure 7, Table 4). Since Q is the equilibrium constant for the disproportion reactions shown in Scheme 2, we used the relationships in eqn 10-12 to obtain the coupling free energies, or free energies of activation, for each interaction. The ∆G values for each interaction were negative, suggesting the interactions have a positive influence on ligand binding (i.e. activation). In striking contrast, the coupling between MgATP and pyruvate was completely abolished in the absence of acetyl-CoA (Q = 1) as evidenced by the lack of influence increasing MgATP concentrations have on the apparent Km for pyruvate (Figure 7).


33

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 50

Figure 7. Variation of the apparent Km for pyruvate as a function of MgATP concentration in the presence (■) or absence () of acetyl-CoA. Apparent Km values for pyruvate were determined by measuring the carboxylation activity at varying concentrations of pyruvate and fixed concentrations in the presence or absence of saturating acetyl-CoA. Estimates of Qab/x were made from the limiting values (dotted lines) of Kib/x and Kiab/x and the magnitude of Qab/x is shown with the vertical arrow. Long dashed lines are the best-fit line of the data to eqn 935. There is no dependence of the apparent Km pyruvate on MgATP concentrations in the absence of acetyl-CoA () and Qab is = 1 (i.e. Kib = Kiba in the absence of acetyl-CoA), indicating a lack of thermodynamic linkage. Parameters derived from the best-fit lines are shown in Table 4.

Effect of acetyl-CoA on the thermodynamic activation parameters. The thermodynamic activation parameters for the SaPC catalyzed carboxylation of pyruvate were determined in the presence and absence of saturating acetyl-CoA (Figure 8, Table 5). Arrhenius plots of –R(ln vi)


34

Page 35 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

in both the presence and absence of acetyl-CoA (Figure 8A) were mostly linear, with only slight deviations at the highest temperatures (> 55 °C), suggesting a single rate-limiting step predominates the reaction over the majority of the temperature range examined (15-55 °C). While the kobs values determined in the presence of acetyl-CoA were significantly higher compared to those determined in the absence of acetyl-CoA, the activation energy for the carboxylation reactions (Ea) were, within experimental error, nearly the same (Table 5).

Figure 8. Temperature dependence of kobs (sec-1) for the SaPC catalyzed carboxylation of pyruvate in the presence (●) and absence (○) of saturating acetyl-CoA. (A) Arrhenius and (B) Eyring plots. kobs is the specific activities determined under saturating conditions for the SaPC catalyzed formation of oxaloacetate at each temperature. Linear portions of the curve were fitted to eqn 13 (A) or eqn 14 (B, dashed lines) to determine thermodynamic activation parameters (Table 5). Activities were determined in triplicate at each temperature. kobs (sec-1) ± std dev from triplicate measurements as a function of temperature are shown in Table S1.


35

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 50

Table 5. Thermodynamic activation parameters for the PC catalyzed carboxylation of pyruvate in the presence and absence of acetyl-CoAa. (+) Acetyl-CoA

(-) Acetyl -CoA

Ea (kJ mol-1)

79 ± 2

78 ± 2

∆H‡ (kJ mol-1)

65 ± 2

68 ± 2

∆S‡ (J mol-1 K-1)

-18.8 ± 0.4

-39.2 ± 0.2

a

Reaction conditions: 100 mM Tricine (pH 7.6), pyruvate (15 mM), MgATP (2.5 mM), MgCl2 (2.5 mM), HCO3- (40 mM), NADH (0.2 mM), malate dehydrogenase (5 U), SaPC (10-75 µg) . kobs was determined in the presence or absence of acetyl-CoA (250 µM) at varying temperatures (15-55 °C) by monitoring the oxidation of NADH to NAD+ using the malate dehydrogenase coupled assay system. All activities were determined in triplicate at each temperature. Reported error is ± std dev for each parameter determined from nonlinear regression.

To determine ∆H‡ and ∆S‡ directly, Eyring plots (Figure 8B) were constructed. Plots of ln vi/T vs. 1/T were linear over the majority of the temperatures examined, with only a slight deviation from linearity at the extreme high and low temperature. Importantly, we observed a significant (~19 J mol-1 K-1) decrease in the activation entropy in the absence of acetyl-CoA as evidenced by the more negative ∆S‡, suggesting that the presence of acetyl-CoA results in a more ordered system.

DISCUSSION Allosteric control of metabolic enzymes is a critical aspect of enzyme function. There remains, however, a poor understanding at the molecular level as to how allostery plays a role in governing enzyme function

62-65

, especially the allosteric regulation of multifunctional enzymes


36

Page 37 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

like PC. The studies presented here contribute to unveiling a functional role for the allosteric activator, acetyl-CoA, in efficiently coordinating catalysis in an α4 PC. Acetyl-CoA and MgATP bind to SaPC prior to the addition of pyruvate. Similar to PC isolated from Rhizobium etli, SaPC exhibits a sigmoidal rate dependence on increasing acetyl-CoA concentrations when all other substrates were saturating. The Hill coefficient of ~2.9 (Table 3) is consistent with multiple acetyl-CoA ligands occupying the allosteric domains during turnover. More importantly, the initial velocity studies indicate an ordered addition of substrates to SaPC, with MgATP and acetyl-CoA binding randomly in the BC and allosteric domains, respectively, prior to the addition of pyruvate in the CT domain (Scheme 3). The proposed kinetic order of addition is also consistent with previous kinetic that show MgATP-cleavage and carboxybiotin formation occurs in the absence of bound pyruvate 24, 52, 66, 67. Acetyl-CoA energetically couples substrates in the BC and CT domains. In the presence of acetyl-CoA, MgATP in the BC domain and pyruvate in the CT domain are thermodynamically coupled. However, the energetic coupling is completely abolished in the absence of acetyl-CoA. Conceptually

59

, these data suggest that protein-mediated interactions between pyruvate and

MgATP occur only in the presence of acetyl-CoA and that in the absence of acetyl-CoA, pyruvate in the CT domain and MgATP in the BC domain are no longer “linked” by this interaction. Considering the relative magnitude of the coupling parameters for acetyl-CoA, pyruvate and MgATP are comparable, we propose that acetyl-CoA, pyruvate and MgATP are thermodynamically linked through a common protein-mediated interaction, underscoring the importance of acetyl-CoA in physically linking the substrates or conformational changes within the two spatially discrete domains. It is possible that protein-mediated interactions responsible for the thermodynamic linkage between acetyl-CoA, pyruvate and MgATP occur via the BCCP


37

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 50

domain, which is in contact with the allosteric, CT and BC domains throughout turnover. In fact, acetyl-CoA may facilitate long-range communication between the BC and CT domains by constraining the movements of the highly flexible BCCP domain, resulting in the decreases in activation entropy

47

observed in the presence of acetyl-CoA in SaPC and the amount of non-

productive MgATP cleavage and premature carboxybiotin decarboxylation. That is, in the absence of acetyl-CoA, the unrestricted movement of the BCCP domain could result in nonproductive carboxybiotin decarboxylation, possibly in either its own BC domain or an empty CT domain active site. Additionally, restriction of the BCCP domain by acetyl-CoA may be the protein-mediated binding event that allows for the thermodynamic link, or energetic coupling, between the CT and BC domains. Differential stabilization of global conformations may give rise to species-specific activation parameters. The development of moderately divergent regulatory mechanisms among species is common and due, in part, to the evolutionary demands placed on the organism

62, 68, 69

. While

both RePC and SaPC catalyze the same reaction through similar, presumably, chemical mechanisms,

there

are

fundamental,

species-specific

differences

in

the

determined

thermodynamic activation parameters. For example, we found that in SaPC, the activation energy (Ea) and enthalpy of activation (∆H‡) are largely unaffected by the presence of acetylCoA while a significant increase (~19-20 J mol-1 K-1) in the ∆S‡ was observed in the absence of acetyl-CoA. In striking contrast, the RePC catalyzed reactions showed significant increases in Ea, ∆H‡, and ∆S‡ in the absence of acetyl-CoA and a corresponding decrease in the Gibbs free energy of activation

17

. Surprisingly, the magnitude of the activation parameters were

significantly greater in the SaPC catalyzed reaction compared to the RePC catalyzed reaction with the exception of ∆S‡, which was ~2-4 times more negative than that determined for SaPC.


38

Page 39 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Importantly, the magnitude of the increases in the entropy of activation in the absence of acetylCoA for both the RePC and SaPC catalyzed reaction were similar. Thermodynamic activation parameters for SaPC are largely independent of acetyl-CoA while those for RePC are dependent on acetyl-CoA. By invoking the rational assumptions that (1) the asymmetric to symmetric global conformational transition is relevant to catalytic turnover (i.e. a “half-the-sites” or obligatory oscillating mechanism) conformers is rate-limiting

24, 30, 52

15, 17, 31

, (2) the transition between the

, thus reflective in the activation energy required to reach the

transition state and, overall, a major thermodynamic driving force of the reaction and (3) the transition between and stabilization of the different conformers is dependent on both speciesspecific residue interactions

10, 14

and the presence or absence of acetyl-CoA

11, 12, 18, 31

, we can

speculate that the differences in thermodynamic parameters may arise from the differential stabilization of global asymmetric or symmetric conformations of the tetramer throughout catalytic turnover (Figure 9). In fact, recent mutagenic studies

15, 17, 31

suggest that variable

inter- and intra-face residue interactions among the domains may not only aid in stabilizing specific conformers of the enzyme (i.e. asymmetric vs. symmetric), but also promote the interconversion between the conformers during turnover.


39

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 50

Figure 9. Proposed model of activation and conformational asymmetric-to-symmetric transitions of SaPC and RePC giving rise to differential thermodynamic parameters. The interconversion between asymmetric and symmetric conformations and relative stability of each conformer could give rise to the difference in thermodynamic parameters. Each ellipsoid represents a single monomer. We presume that

14-17

, SaPC (blue) and RePC (green) are most

likely stable in a symmetric and an asymmetric conformer, respectively (far left), in the presence of absence of acetyl-CoA (red circle). In the asymmetric state for either PC, only two of the four acetyl-CoA binding sites are available9, while in the symmetric state all four can be occupied. Differential stabilization of the respective conformers arises from interdomain interactions between non-conserved amino acid residues. Further, the energy requirements for breaking or forming of these contacts (i.e. H-bond interactions, salt bridges) in SaPC is different than in RePC, since the amino acid residue interactions are different, thus additionally contributing to


40

Page 41 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

the thermodynamic differences. In SaPC, transition to the asymmetric conformer with the BCCP domain associated with the BC domain (Asym-BC) would be the most thermodynamically uphill transition while the transitions from the Asym-BC to the Sym-CT conformer would be the most energetically costly for RePC. Transtion for SaPC back to the symmetric conformer would be facilitated by the re-establishment of interdomain contacts between the non-conserved amino acid residues. Obligatory oscillating catalysis, where catalytic activity occurs on alternate, opposing faces of the tetramer, is represented in the far right panel, however, obligate oscillation has not been definitively defined.

Cyro-EM studies of SaPC actively undergoing turnover have revealed a wealth of information regarding the possible location of the BCCP domain and global architecture of the enzyme during catalysis15. Importantly, it suggests that both RePC and SaPC undergo symmetric-toasymmetric transitions during catalysis. Based on these studies, to initiate catalysis, both RePC and SaPC would need to adopt the Asym-BC conformation with the BCCP domain inserted into the BC domain active site (Figure 9). If we presume that the most stable conformation of the SaPC tetramer is a symmetric conformer14-17, achieving the Asym-BC conformation would require a significant amount of thermodynamic energy compared to the relatively thermodynamically favorable step of transitioning between the Asym-BC to Sym-CT conformation. Assuming that acetyl-CoA occupies all four allosteric domains14, the transition would require the loss of two acetyl-CoA molecules and, presumably, be independent of acetylCoA binding. The increased activation energy and enthalpy for the SaPC catalyzed reaction is then attributable to the drastic conformational changes in converting between the symmetrical, energetically favorable conformation to the Asym-BC conformation (Figure 9). Further, the


41

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 50

SaPC Asym-BC to Sym-CT transition would be, in general, more energetically favorable than the same transition in RePC. Because of the favorable energetics and enthalpy-entropy compensation mechanisms, this line of reasoning would also support the smaller ∆S‡ observed for SaPC compared to RePC. In contrast, considering the asymmetric nature of RePC, the small conformational adjustment of moving the BCCP from the exo site to the BC domain to initiate catalysis is not relatively costly in terms of thermodynamic penalties13, 27. On the other hand, transitioning from the AsymBC RePC conformer to the Sym-CT RePC conformer is expected to be not only thermodynamically costly relative to the initial transition, but would presumably be dependent on acetyl-CoA. Our model would then suggest the majority of the Ea for the RePC catalyzed reaction is due to the Asym-BC to Sym-CT transition and, consistent with the observed activation parameters, most likely be acetyl-CoA dependent17. Additionally, the differential stabilization of the respective conformers arises from interdomain interactions between nonconserved amino acid residues. The energy requirements for breaking or forming of these contacts (i.e. H-bond interactions, salt bridges) in SaPC is different than in RePC, since the amino acid residue interactions are different, thus additionally contributing to the observed differences in thermodynamic parameters17. While the idea of PC possessing a half-the-sites activity or obligatory oscillating catalytic mechanism has arisen from mostly structural conjecture, the variability in inter and intra-face residue interactions between the allosteric and BC/CT domains has been shown to have differential effects on PC tetramerization

14, 70, 71

and

acetyl-CoA activation 31, suggesting there may be fundamental species-specific differences in the thermodynamics governing the allosteric mechanism.


42

Page 43 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

In summary, our results show that acetyl-CoA kinetically and energetically couples the substrates of the BC ad CT domains. In addition, our data shows that the thermodynamic activation parameters for the SaPC catalyzed reactions were largely independent of acetyl-CoA and differing from those determined for RePC. One way acetyl-CoA may facilitate long-range communication between the BC and CT domains could be by influencing the movements of the mobile BCCP domain or selectively promoting and stabilizing conformational interconversions of the enzyme. Constraining the movements of the highly flexible BCCP domain would result in the decreases in activation entropy

47

observed in the presence of acetyl-CoA while stabilizing

specific conformational of the enzyme would lend itself to species specific differences in activation parameters. In this way, acetyl-CoA is pivotal to synchronizing global conformational changes and domain movements in the tetramer with catalytic activity, since the intra and interfacial contacts are rearranged as the enzyme transitions from the asymmetric to symmetric conformer. Taken as a whole, our results contribute to defining the role of acetyl-CoA in the activation of α4 PCs from a variety of sources.


43

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 50

ASSOCIATED CONTENT Supporting Information. Supporting information including additional kinetic data and statistical analysis (Figs S1-S5, Table S1-S5) 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: [email protected]; Address: East Carolina Heart Institute, 115 Heart Drive, Greenville, NC, 27834. Ph: 252-744-5609. 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 Funding for this work was provided by the Brody Brothers Foundation (Grant 225132 to TNZ and SRS), the East Carolina University Office of Research and Graduate Studies (to TNZ) and the National Heart, Lung and Blood Institute at the National Institutes of Health (Grant R01 HL123647 to SRS). ACKNOWLEDGMENT We thank Dr. Liang Tong for providing the SaPC expression plasmid.

ABBREVIATIONS


44

Page 45 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Acetyl-CoA, acetyl-Coenzyme A; BC, biotin carboxylase; BCCP, biotin carboxyl carrier protein; CT, carboxyl transferase; Sa, Staphylococcus aureus; Re, Rhizobium etli, PC, pyruvate carboxylase.

REFERENCES [1] Jitrapakdee, S., Wutthisathapornchai, A., Wallace, J. C., and MacDonald, M. J. (2010) Regulation of insulin secretion: role of mitochondrial signalling, Diabetologia 53, 10191032. [2] Vongpipatana, T., Wutthisathapornchai, A., and Jitrapakdee, S. (2013) Identification of transcriptional factors that bind to the glucose-responsive elements of the pyruvate carboxylase gene in pancreatic beta cells, In Signaling and Transcriptional Control in Endocrine Systems, pp SAT-405-SAT-405, Endocrine Society. [3] 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. [4] Phannasil, P., Thuwajit, C., Warnnissorn, M., Wallace, J. C., MacDonald, M. J., and Jitrapakdee, S. (2015) Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells, PLoS one 10, e0129848. [5] Schär, J., Stoll, R., Schauer, K., Loeffler, D. I., Eylert, E., Joseph, B., Eisenreich, W., Fuchs, T. M., and Goebel, W. (2010) Pyruvate carboxylase plays a crucial role in carbon metabolism of extra-and intracellularly replicating Listeria monocytogenes, J. Bacteriol. 192, 1774-1784. [6] Eylert, E., Schär, J., Mertins, S., Stoll, R., Bacher, A., Goebel, W., and Eisenreich, W. (2008) Carbon metabolism of Listeria monocytogenes growing inside macrophages, Mol. Microbiol. 69, 1008-1017. [7] Hain, T., Chatterjee, S. S., Ghai, R., Kuenne, C. T., Billion, A., Steinweg, C., Domann, E., Kärst, U., Jänsch, L., and Wehland, J. (2007) Pathogenomics of Listeria spp, Int. J. Med. Microbiol. 297, 541-557. [8] Benton, B. M., Zhang, J., Bond, S., Pope, C., Christian, T., Lee, L., Winterberg, K. M., Schmid, M. B., and Buysse, J. M. (2004) Large-scale identification of genes required for full virulence of Staphylococcus aureus, J. Bacteriol. 186, 8478-8489. [9] 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. [10] Xiang, S., and Tong, L. (2008) Crystal structures of human and Staphylococcus aureus pyruvate carboxylase and molecular insights into the carboxyltransfer reaction, Nat. Struct. Mol. Biol. 15, 295-302. [11] Adina-Zada, A., Zeczycki, T. N., and Attwood, P. V. (2012) Regulation of the structure and activity of pyruvate carboxylase by acetyl CoA, Arch. Biochem. Biophys. 519, 118-130.


45

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 50

[12] Adina-Zada, A., Zeczycki, T. N., St Maurice, M., Jitrapakdee, S., Cleland, W. W., and Attwood, P. V. (2012) Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA, Biochem. Soc. Trans. 40, 567-572. [13] 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. [14] 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. [15] 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. [16] Lasso, G., Yu, L. P. C., Gil, D., Xiang, S., Tong, L., and Valle, M. (2010) Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase, Structure 18, 1300-1310. [17] Sirithanakorn, C., Jitrapakdee, S., and Attwood, P. V. (2016) Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA, Biochemistry 55, 4220-4228. [18] 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. [19] Adina-Zada, A., Hazra, R., Sereeruk, C., Jitrapakdee, S., Zeczycki, T. N., Maurice, M. S., Cleland, W. W., Wallace, J. C., and Attwood, P. V. (2011) Probing the allosteric activation of pyruvate carboxylase using 2′, 3′-O-(2, 4, 6-trinitrophenyl) adenosine 5′triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA, Arch. Biochem. Biophys. 509, 117-126. [20] 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. [21] 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. [22] 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. [23] 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. [24] 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, Ann. N. Y. Acad. Sci. 447, 169-188.


46

Page 47 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[25] 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. [26] 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. [27] 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. [28] 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. [29] Legge, G. B., Branson, J. P., and Attwood, P. V. (1996) Effects of acetyl CoA on the presteady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase, Biochemistry 35, 3849-3856. [30] Attwood, P. V., Wallace, J. C., and Keech, D. B. (1984) The carboxybiotin complex of pyruvate carboxylase. A kinetic analysis of the effects of Mg2+ ions on its stability and on its reaction with pyruvate, Biochem. J 219, 243-251. [31] Choosangtong, K., Sirithanakorn, C., Adina-Zada, A., Wallace, J. C., Jitrapakdee, S., and Attwood, P. V. (2015) Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation, FEBS Letters 589, 2073-2079. [32] Westerhold, L. E., Adams, S. L., Bergman, H. L., and Zeczycki, T. N. (2016) Pyruvate Occupancy in the Carboxyl Transferase Domain of Pyruvate Carboxylase Facilitates Product Release from the Biotin Carboxylase Domain through an Intermolecular Mechanism, Biochemistry 55, 3447-3460. [33] Fenton, A. W. (2008) Allostery: an illustrated definition for the ‘second secret of life’, Trends Biochem. Sci. 33, 420-425. [34] Symcox, M. M., and Reinhart, G. D. (1992) A steady-state kinetic method for the verification of the rapid-equilibrium assumption in allosteric enzymes, Anal. Biochem. 206, 394-399. [35] Reinhart, G. D. (2004) Quantitative analysis and interpretation of allosteric behavior, Methods Enzymol. 380, 187-203. [36] Reinhart, G. D. (1983) The determination of thermodynamic allosteric parameters of an enzyme undergoing steady-state turnover, Archives of biochemistry and biophysics 224, 389-401. [37] Motulsky, H., and Christopoulos, A. (2004) Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting, OUP USA. [38] Dalziel, K. (1957) Initial steady state velocities in the evaluation of enzyme-coenzymesubstrate reaction mechanisms, Acta Chem. Scand. 11, 706-723. [39] Cleland, W. W. (1970) 1 Steady State Kinetics, In The Enzymes (Paul, D. B., Ed.), pp 1-65, Academic Press. [40] Schmider, J., Greenblatt, D.J. , Harmatz, J.S., and Shader, R. I. (1996) Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N‐demethylation, Br. J. Clin. Pharmacol. 41, 593-604.


47

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 50

[41] Segel, I. Enzyme Kinetics: BehaViour and analysis of rapid equilibrium and steady-state enzyme systems, 1993, New York: John Wiley & Sons. [42] Reinhart, G. D. (1988) Linked-function origins of cooperativity in a symmetrical dimer, Biophy. Chem. 30, 159-172. [43] Weber, G. (1975) Energetics of ligand binding to proteins, Adv. Protein Chem. 29, 68. [44] Weber, G. (1972) Ligand binding and internal equilibiums in proteins, Biochemistry 11, 864-878. [45] Ortigosa, A. D., Kimmel, J. L., and Reinhart, G. D. (2004) Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus, Biochemistry 43, 577-586. [46] Johnson, J. L., and Reinhart, G. D. (1997) Failure of a two-state model to describe the influence of phospho (enol) pyruvate on phosphofructokinase from Escherichia coli, Biochemistry 36, 12814-12822. [47] Reinhart, G. D., Hartleip, S. B., and Symcox, M. M. (1989) Role of coupling entropy in establishing the nature and magnitude of allosteric response, PNAS 86, 4032-4036. [48] Ashman, L. K., Keech, D. B., Wallace, J. C., and Nielsen, J. (1972) Sheep Kidney Pyruvate Carboxylase: Studies On Its Activation By Acetyl Coenzyme A And Characteristics Of Its Acetyl Coenzyme A Independent Reaction, J. Biol. Chem. 247, 5818-5824. [49] Easterbrook-Smith, S. B., Wallace, J. C., and Keech, D. B. (1978) A reappraisal of the reaction pathway of pyruvate carboxylase, Biochem. J 169, 225-228. [50] 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. [51] Cleland, W. (1963) The kinetics of enzyme-catalyzed reactions with two or more substrates or products: III. Prediction of initial velocity and inhibition patterns by inspection, Biochim. Biophys. Acta 67, 188-196. [52] Attwood, P., and Wallace, J. (1986) The carboxybiotin complex of chicken liver pyruvate carboxylase. A kinetic analysis of the effects of acetyl-CoA, Mg2+ ions and temperature on its stability and on its reaction with 2-oxobutyrate, Biochem. J 235, 359-364. [53] Barden, R. E., Fung, C.-H., Utter, M. F., and Scrutton, M. C. (1972) Pyruvate Carboxylase from Chicken Liver: Steady State Kinetic Studies Indicate A" Two-Site" Ping-Pong Mechanism, J. Biol. Chem. 247, 1323-1333. [54] 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. [55] Warren, G. B., and Tipton, K. F. (1974) Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate, Biochem. J.139, 311-320. [56] Ashman, L., and Keech, D. (1975) Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation, J. Biolo. Chem. 250, 1421. [57] Libor, S. M., Sundaram, T. K., and Scrutton, M. C. (1978) Pyruvate carboxylase from a thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator, Biochem. J.l 169, 543-558. [58] Wyman, J. (1975) A group of thermodynamic potentials applicable to ligand binding by a polyfunctional macromolecule, PNAS 72, 1464-1468. [59] Wyman, J. (1967) Allosteric Linkage, J. Am. Chem. 89, 2202-2218.


48

Page 49 of 50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[60] Wyman Jr, J. (1964) Linked Functions and Reciprocal Effects in Hemoglobin: A Second Look, In Advances in Protein Chemistry (C.B. Anfinsen, M. L. A. J. T. E., and Frederic, M. R., Eds.), pp 223-286, Academic Press. [61] Braxton, B., Mullins, L. S., Raushel, F. M., and Reinhart, G. D. (1996) Allosteric effects of carbamoyl phosphate synthetase from Escherichia coli are entropy-driven, Biochemistry 35, 11918-11924. [62] Nussinov, R., and Tsai, C.-J. (2015) Allostery without a conformational change? Revisiting the paradigm, Curr. Opin. Struct. Biol. 30, 17-24. [63] Nussinov, R., Ma, B., and Tsai, C.-J. (2014) Multiple conformational selection and induced fit events take place in allosteric propagation, Biophys. Chem. 186, 22-30. [64] Tsai, C.-J., and Nussinov, R. (2014) A Unified View of “How Allostery Works”, PLoS Comput. Biol. 10, e1003394. [65] Tsai, C.-J., Del Sol, A., and Nussinov, R. (2009) Protein allostery, signal transmission and dynamics: a classification scheme of allosteric mechanisms, Mol. BioSyst. 5, 207-216. [66] WARREN, G. B., and TIPTON, K. F. (1974) The Role of Acetyl‐CoA in the Reaction Pathway of Pig‐Liver Pyruvate Carboxylase, Eur. J. Biochem. 47, 549-554. [67] Attwood, P. V., and Graneri, B. D. (1992) Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate, Biochem.J 287, 1011-1017. [68] Goodey, N. M., and Benkovic, S. J. (2008) Allosteric regulation and catalysis emerge via a common route, Nat. Chem. Biol. 4, 474-482. [69] Nussinov, R., and Tsai, C.-J. (2013) Allostery in Disease and in Drug Discovery, Cell 153, 293-305. [70] Yu, L. P., Chou, C.-Y., Choi, P. H., and Tong, L. (2013) Characterizing the Importance of the Biotin Carboxylase Domain Dimer for Staphylococcus aureus Pyruvate Carboxylase Catalysis, Biochemistry 52, 488-496. [71] Shen, Y., Chou, C.-Y., Chang, G.-G., and Tong, L. (2006) Is dimerization required for the catalytic activity of bacterial biotin carboxylase?, Mol. Cell 22, 807-818.


49

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 50

TABLE OF CONTENTS GRAPHIC-FOR TOC USE ONLY


50

Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of

Recommend Documents