Investigation of the Roles of Allosteric Domain Arginine, Aspartate

Jul 5, 2016 - and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in. Relation to Its Activation by Acetyl CoA. Chaiyos Sirithanakorn,. †...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/biochemistry

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 Chaiyos Sirithanakorn,† Sarawut Jitrapakdee,*,† and Paul V. Attwood*,‡ †

Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand School of Chemistry and Biochemistry, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia

‡

ABSTRACT: The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using sitedirected mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.

P

as a swinging arm carrying the carboxyl group from the BC domain to the CT domain. The allosteric domain has a role in the stabilization of tetrameric structure and activation of the catalytic reaction when the allosteric activator, acetyl CoA, is bound (Figure 1). In the asymmetric RePC tetramer structure, one pair of subunits that was configured for intersubunit catalysis had ethyl CoA (an acetyl CoA analogue) bound (Figure 1b), while the other pair of subunits did not.9 However, a later structure obtained in the absence of either ethyl CoA or acetyl CoA showed that this asymmetry is not due to activator binding but is an inherent property of the RePC tetramer.8 In addition, the Hill coefficient for the cooperative activation of RePC by acetyl CoA is >2, suggesting that all the allosteric activator sites in the tetramers may be occupied during catalysis. Recently, Lasso et al.7 demonstrated that in S. aureus PC (SaPC) there are transitions between symmetrical and asymmetrical configurations of the tetramer, concomitant with movements of BCCP domains of the enzyme during catalysis. These authors also provided evidence that supports the model of half-of-sites reactivity for PC, in which only one pair of subunits is configured to support catalysis at any one

yruvate carboxylase (PC, EC 6.4.1.1.) is a biotin-dependent carboxylase, which catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. This is a crucial anaplerotic reaction that replenishes tricarboxylic acid cycle intermediates upon cataplerosis.1 In some pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus, PC plays a role with respect to replication and virulence.2 In mammals, it is involved in several intermediary metabolic pathways, including gluconeogenesis in liver and kidney, lipogenesis in adipose tissue, glucose-induced insulin secretion in pancreatic β cells, and neurotransmitter synthesis in astrocytes. In humans, the aberrant expression of PC is associated with metabolic syndromes3,4 and various types of cancer.5,6 Recently, a number of structures of PC of the major homotetrameric (α4) form have been determined.7−12 Each subunit consists of four functional domains, the biotin carboxylase (BC), carboxyl transferase (CT), biotin carboxyl carrier protein (BCCP), and allosteric domains (Figure 1a). The bicarbonate-dependent MgATP cleavage reaction occurs in the BC domain, which results in the carboxylation of biotin, whereas the carboxyl transfer from carboxybiotin to pyruvate, forming oxaloacetate, occurs in the CT domain. The BCCP domain, which contains the covalently bound biotin, functions © XXXX American Chemical Society

Received: May 31, 2016

A

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

between 44 and 55 Å from the CT domain active site and have no direct roles in the well-characterized mechanisms of catalysis of reactions in the BC domain15 or the CT domain.14,17 It is thus evident that the activating effects on catalysis produced by mutation of these residues are allosteric in nature. Here, we investigate the functional role of the remaining residue in the allosteric domain of RePC that directly interacts with acetyl CoA, Arg429 (Figure 2). In addition, the roles of Asp420,

Figure 1. (a) Domain architecture of RePC. The BC, CT, BCCP, and allosteric domains are colored blue, yellow, red, and green, respectively. The catalytic reaction that occurs in each domain is shown below the corresponding domain structure. (b) Cartoon showing the face of the RePC tetramer with subunits that have ethyl CoA (an analogue of acetyl CoA) bound and are configured for intersubunit catalysis as shown. Figures reproduced with permission from ref 9. Copyright 2007 American Association for the Advancement of Science.

time, and that in the course of catalysis, this activity alternates between pairs of subunits on opposite faces of the tetramer.7 The catalytic mechanism of PC has previously been described.13−17 In brief, enzyme-bound biotin is carboxylated in the BC domain via the formation of the carboxyphosphate intermediate upon cleavage of MgATP. The BCCP domain then swings from the BC domain of its own subunit to the CT domain of the opposing subunit of the same dimer pair, where the carboxyl group is transferred to pyruvate to form oxaloacetate (Figure 1b). There is a great deal of variation in the activation of α4-type PCs by acetyl CoA. Some are not activated at all, while others show variations in activity in the presence and absence of acetyl CoA, the Ka for activation, and the degree of activation.18 The main locus of action of acetyl CoA appears to lie in the BC domain where MgATP cleavage and biotin carboxylation occur.19−21 Previous studies examined the roles of some of the residues that are involved in the binding of acetyl CoA and its activation of RePC.22,23 In addition, the mutations of some of these residues were gain-of-activity mutations that were found to result in activation of the enzyme in the absence of acetyl CoA, primarily affecting reactions in the BC domain. These residues and the residues examined in the work presented here are between 27 and 47 Å from the BC domain active site and

Figure 2. (a) Residues surrounding ethyl CoA (an acetyl CoA analogue) in the allosteric domain of RePC (PDB entry 2QF7). Roles of Arg427, Arg472, and Arg469 (gray) have been previously characterized.30,31 Asp420, Arg424, Glu425, and Arg429 (colored by atom) are previously uncharacterized residues in the acetyl CoA binding site and were selected for investigation in this work. Arg20 and Lys45 (light blue) are residues in an adjacent subunit in the RePC tetramer. Dashed lines indicate potential interactions between residues, and distances between residues relevant to this work are indicated. (b) Amino acid sequence alignment of PC from various species: mPC, mouse PC; HuPC, human PC; BsPC, Bacillus stearothermophilus PC; SaPC, S. aureus PC; RePC, Rhizobium etli PC.

Arg424, and Glu425 are examined. These three residues form interactions with each other and, in the case of Glu425, with residues from another subunit in the RePC tetramer. These residues have no direct contact with bound acetyl CoA but do interact with Arg427 that has a very important role in acetyl CoA binding22 (Figure 2a). In addition, the mutation of Arg427 to serine not only inhibited acetyl CoA binding but also inhibited bicarbonate-dependent MgATP cleavage in the presence and absence of acetyl CoA, phosphorylation of B

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry MgADP by carbamoyl phosphate, and biotin carboxylation.22 Thus, Adina-Zada et al.22 proposed that Arg427 is important for the correct positioning of the BCCP domain (and hence the covalently bound biotin) in the BC domain, not through direct contacts, however, because Arg427 is 40 Å from the BC domain. In this work, we have performed site-directed mutagenesis on these residues and measured the effect on catalysis, allosteric activation by acetyl CoA, and, in the case of Arg424, how the mutation of this residue to serine affects the thermodynamic activation parameters for catalysis in the absence of acetyl CoA.

k′cat = k 0cat + kcat /[1 + (K a /[acetyl CoA])h ]

where k′cat is the measured catalytic rate constant at various concentrations of acetyl CoA, k0cat is the catalytic rate constant in the absence of acetyl CoA, kcat is the catalytic rate constant of the acetyl CoA-dependent enzymic activity, Ka is the activation constant for acetyl CoA, and h is the Hill coefficient of activation. Values of k′cat were calculated by dividing measured reaction velocities by the enzymic biotin concentration used in each assay. Bicarbonate-Dependent MgATP Cleavage Activity Assay. The bicarbonate-dependent MgATP cleavage reactions in the absence or presence of saturating concentrations of acetyl CoA were assessed by a coupled spectrophotometric method in which pyruvate kinase and lactate dehydrogenase were used as coupling enzymes to measure MgADP release. The reactions were performed at 30 °C in a 1 mL reaction volume containing 100 mM Tris-HCl (pH 7.8), 20 mM NaHCO3, 6 mM MgCl2, 1 mM MgATP, 5 mM phosphoenolpyruvate, 0.22 mM NADH, 5 units of pyruvate kinase, and 4 units of lactate dehydrogenase and purified RePC enzyme. The kcat value was calculated from initial velocity divided by the enzyme biotin concentration used in each assay. Oxamate-Induced Oxaloacetate Decarboxylation Activity Assay. The oxamate-induced oxaloacetate decarboxylation activities in the absence or presence of saturating concentrations of acetyl CoA were determined by a coupled assay in which pyruvate is converted to lactate by lactate dehydrogenase concomitant with oxidation of NADH. All assays were performed at 30 °C in a 1 mL reaction mixture containing 100 mM Tris-HCl (pH 7.8), 20 μM oxaloacetate, 1 mM oxamate, 0.22 mM NADH, and 4 units of lactate dehydrogenase. The kcat value was calculated from initial velocity divided by the enzyme biotin concentration used in each assay. Coupling between Oxaloacetate Formation and Pi Release in the Pyruvate Carboxylation Reaction. To determine the effect of mutation of Arg424 on the coupling between oxaloacetate formation and Pi release in the pyruvate carboxylation reaction in the presence or absence of saturating concentrations of acetyl CoA, the rates of oxaloacetate formation and Pi release catalyzed by the R424S mutant were measured and compared with those of the wild-type enzyme. The rates of Pi release and the oxaloacetate formation were measured as described previously.26 Coupling between oxaloacetate formation and Pi release was calculated as the ratio of the rate of these two reactions in three separate measurements. The ratio is shown as the mean ± the standard deviation of these measurements. Temperature Dependence of k0cat and kcat of WildType RePC and k0cat of the R424S Mutant for the Pyruvate Carboxylation Reaction. Assays were performed at 15, 20, 25, 30, 35, and 40 °C. The reactions were performed as described above, and the mixtures were equilibrated for 10 min to reach the temperature prior to the addition of the enzyme. Reactions with wild-type RePC were performed in the presence of a saturating concentration of acetyl CoA or in its absence, while those for R424S were performed in the absence of acetyl CoA. The modified Arrhenius plot was obtained for each data set by plotting 1/T (K−1) against R ln kcat. The activation energy (Ea) was obtained by linear least-squares regression analysis of the data. The activation parameters were calculated at 30 °C. The activation enthalpy (ΔH⧧) was

■

EXPERIMENTAL PROCEDURES RePC Mutant Construction. D420A, R424S, E425A, E425Q, R429S, and R429K RePC mutants were created on a 1.4 kb SacII−XhoI DNA fragment of RePC gene encoding the BC domain using site-directed mutagenesis. The primers used for generating the mutants are listed in Table 1. The putative Table 1. Oligonucleotide Primers Used to Generate Rhizobium etli PC Mutants mutant construct D420A R424S E425A E425Q R429S R429K

forward primer

reverse primer

5′ ATT TCC CGC ATG GCC CGG GCG CTG CGC 3′ 5′ GAC CGG GCG CTG AGC GAA TTC CGC ATG 3′ 5′ CGG GCG CTG CGC GCA TTC CGC ATC CGT 3′ 5′ CGG GCG CTG CGC CAA TTC CGC ATC C 3′ 5′ ATT CCG CAT CAG TGG CGT CGC 3′ 5′ GAA TTC CGC ATC AAA GGC GTC GCC ACC 3′

5′ GCG CAG CGC CCG GGC CAT GCG GGA AAT 3′ 5′ CAT GCG GAA TTC GCT CAG CGC CCG GTC 3′ 5′ ACG GAT GCG GAA TGC GCG CAG CGC CCG 3′ 5′ GGA TGC GGA ATT GGC GCA GCG CCC G 3′ 5′ GCG ACG CCA CTG ATG CGG AAT 3′ 5′ GGT GGC GAC GCC TTT GAT GCG GAA TTC 3′

(i)

mutagenic clones were verified by automated DNA sequencing (Macrogen). The equivalent fragment of wild-type RePC was replaced by the mutagenized fragments in pET17b vector by digesting with SacII and XhoI. Expression and Purification of Wild-Type RePC and Its Mutants. pET17b containing wild-type RePC or its mutants was cotransformed with the pCY216 plasmid encoding bacterial biotin protein ligase24 into Escherichia coli BL21(DE3). RePC was expressed and purified as described previously.22,23 Purified RePC was stored at −80 °C in storage buffer containing 30% (v/v) glycerol, 100 mM Tris-HCl (pH 7.8), and 1 mM DTE. Determination of the Biotin Content of RePC. The RePC enzymes were digested in triplicate with 2% (w/v) chymotrypsin in 0.2 M KH2PO4 (pH 7.2) at 37 °C for 24 h and subsequently digested with 5% (w/v) protease from Steptomyces griseus at 37 °C for 48 h. The spectrophotometric dye-displacement biotin assay was performed as described by Green.25 Pyruvate Carboxylation Activity Assay. The pyruvate carboxylating activity in the absence or presence of acetyl CoA was determined by the coupled spectrophotometric method to measure oxaloacetate release.13 The assays were performed at 30 °C in 1 mL of a reaction mixture containing 100 mM TrisHCl (pH 7.8), 20 mM NaHCO3, 6 mM MgCl2, 1 mM MgATP, 0.22 mM NADH, 10 mM sodium pyruvate, and 5 units of malate dehydrogenase, with various concentrations of acetyl CoA. The data were analyzed by nonlinear regression fits as shown in eq i C

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

Figure 3. Acetyl CoA activation of the pyruvate carboxylation reaction of wild-type RePC and its mutants. Solid lines represent nonlinear leastsquares fits of the data to eq i.

calculated from Ea, the activation free energy (ΔG⧧) using the value of either k0cat or kcat measured at 30 °C, and the activation entropy (ΔS⧧) using the relationship ΔG⧧ = ΔH⧧ − TΔS⧧.

substitutions of these residues reduce the affinity of acetyl CoA for the enzyme, most severely in the R429S mutant. However, the kcat values of D420A, R424S, R429S, and R429K mutants were 43, 74, 47, and 43% of that of wild-type RePC, respectively, and show that these mutations did affect the ability of acetyl CoA to activate catalysis. However, the k0cat values of these four mutants were approximately 1.5-, 2-, 2.8-, and 3.2fold greater than that of the wild-type enzyme, respectively, indicating that these substitutions facilitate the acetyl CoAindependent reaction. Furthermore, the Hill coefficient (h) of R424S was increased to 3.24, whereas it was decreased to 1.34 and 1.68 in R429S and R429K, respectively, compared to that of the wild-type enzyme (2.27 ± 0.41), indicating that Arg424 and Arg429 residues mediate the cooperativity of acetyl CoA activation. However, this appears not to be the case for D420A (h = 2.33 ± 0.24). Effects of D420A, R424S, and R429S Mutations on the Bicarbonate-Dependent MgATP Cleavage and Oxamate-Induced Oxaloacetate Decarboxylation Partial Reactions. Previous studies have shown that acetyl CoA mainly activates the MgATP cleavage reaction in the BC domain but does not have a strong effect on pyruvate carboxylation in the CT domain.19−21,27 The catalytic rate constants in the presence (kcat) of saturating acetyl CoA or its absence (k0cat) for wild-type- and mutant-catalyzed bicarbonatedependent MgATP cleavage reaction (in the absence of pyruvate) and oxamate-induced oxaloacetate decarboxylation reaction (reverse CT domain reaction) were determined and are listed in Table 3. The k0cat values for the bicarbonate-dependent MgATP cleavage reaction of D420A, R424S, and R429S show that these mutants have increased activity in the absence of acetyl CoA (approximately 4.7-, 15.5-, and 12.6-fold, respectively), whereas k0cat of R429K was not significantly different from that of wildtype RePC. However, while kcat values for R424S and R429S were 2- and 1.4- fold greater than that for wild-type RePC,

■

RESULTS Substitutions of Glu425. E425A and E425Q failed to express in E. coli BL21(DE3), suggesting that mutations at this residue may disrupt the structure of the enzyme, thus preventing further studies of this residue. Effects of Substitutions of Asp420, Arg424, and Arg429 on the Kinetic Parameters of Acetyl CoA Activation of the Pyruvate Carboxylation Reaction. To examine the involvement of Asp420, Arg424, and Arg429 residues in the acetyl CoA-dependent and -independent pyruvate carboxylating activities, D420A, R424S, R429S, and R429K mutants were generated. The acetyl CoA-induced activation of the pyruvate carboxylation reaction of the wild type and other mutants was investigated, and the data are shown in Figure 3 and Table 2. The Ka values for acetyl CoA activation of D420A, R424S, R429S, and R429K were approximately 2-, 7.5-, 100-, and 4-fold greater than that of wild-type RePC, respectively. These results indicate that Table 2. Kinetic Parameters of Acetyl CoA Activation of the Pyruvate Carboxylation Reactiona RePC wild type D420A R424S R429S R429K

k0cat (s−1)

kcat (s−1)

Ka acetyl CoA (μM)

Hill coefficient

0.26 ± 0.03

13.30 ± 0.94

9.3 ± 0.9

2.27 ± 0.41

0.37 0.51 0.72 0.84

± ± ± ±

0.03 0.15 0.05 0.08

5.73 9.80 6.19 5.67

± ± ± ±

0.19 0.33 0.82 0.21

18.5 69.2 962 37.1

± ± ± ±

0.9 2.6 209 2.4

2.33 3.24 1.34 1.68

± ± ± ±

0.24 0.38 0.16 0.14

a Estimates of parameters were derived from fits of the data to the equation in ref 29, and the errors are standard errors of the estimate.

D

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry Table 3. Effect of Mutations on Partial Reactions in the Absence or Presence of Acetyl CoAa bicarbonate-dependent MgATP cleavage reaction RePC wild type D420A R424S R429S R429K a

no acetyl CoA k0cat (s−1) 0.008 0.037 0.124 0.101 0.006

± ± ± ± ±

0.002 0.001 0.002 0.003 0.005

oxamate-induced oxaloacetate decarboxylation reaction

saturated acetyl CoA kcat (s−1) 0.072 0.049 0.139 0.104 0.024

± ± ± ± ±

0.006 0.002 0.005 0.002 0.002

no acetyl CoA k0cat (s−1) 0.036 0.071 0.049 0.125 0.049

± ± ± ± ±

0.015 0.005 0.001 0.001 0.010

saturated acetyl CoA kcat (s−1) 0.041 0.064 0.052 0.123 0.033

± ± ± ± ±

0.017 0.003 0.008 0.001 0.004

Values of the parameters shown are the means of three measurements ± the standard deviation.

respectively, the values of kcat for D420A and R429K were only 68 and 33% of the kcat for wild-type RePC, respectively. On the other hand, most mutants had values of k0cat and kcat that were not greatly different from those of wild-type RePC in the oxamate-induced oxaloacetate decarboxylation reaction. Only D420A and R429S had values of k0cat that were increased 2- and 3.5-fold, respectively, while only R429S had an increased value for kcat (3-fold) (Table 3). Effect of Substitution of Arg424 with Serine on the Efficiency of Coupling between MgATP Cleavage and Pyruvate Carboxylation in the Absence of Acetyl CoA. The coupling between MgATP cleavage in the BC domain and carboxylation of pyruvate in the CT domain was investigated by measuring the rate of oxaloacetate formation and Pi release in the absence and presence of a saturating acetyl CoA concentration under identical reaction conditions. The ratios of the rate of oxaloacetate formation to that of Pi release in the presence and absence of acetyl CoA were then calculated. In the absence of acetyl CoA, the ratios were 0.70 ± 0.04 and 0.72 ± 0.01 for wild-type RePC and R424S, respectively, while in the presence of acetyl CoA, the values were 1.05 ± 0.07 and 1.10 ± 0.10, respectively. Thus, there was no significant effect of the mutation of Arg424 on the coupling efficiency in the presence or absence of acetyl CoA. Effect of Acetyl CoA and Substitution of Arg424 with Serine on the Thermodynamic Activation Parameters for Pyruvate Carboxylation. The thermodynamic activation parameters of wild-type RePC were determined in the presence and absence of a saturating concentration acetyl CoA, while those of R424S were determined in its absence, using Arrhenius plots (Figure 4 and Table 4). The linearity of the plots in Figure 4 suggests that a single rate-limiting step is being monitored throughout the temperature range. Clearly, the catalytic rate constants for the reaction of wild-type RePC in

Table 4. Thermodynamic Activation Parameters for the Pyruvate Carboxylation Reaction of Wild-Type RePC (WT) in the Presence and Absence of Acetyl CoA and R424S in Its Absence

activation energy Ea (kJ mol−1) ΔH⧧ (kJ mol−1)a ΔS⧧ (kJ mol−1 K−1)a ΔG⧧ (kJ mol−1)a a

WT (without acetyl CoA)

R424S (without acetyl CoA)

WT (with acetyl CoA)

52.4 ± 1.6

39.1 ± 3.8

46.1 ± 2.1

49.9 −0.092 77.7

36.6 −0.123 74.0

43.6 −0.079 67.7

ΔH⧧, ΔS⧧, and ΔG⧧ values were calculated at 30 °C.

the presence of acetyl CoA were greater than those in its absence, while those of R424S in the absence of acetyl CoA were greater than the corresponding values for wild-type RePC, across the entire temperature range. The activation energy for the reaction in wild-type RePC was ∼6 kJ mol−1 smaller in the presence of acetyl CoA than in its absence. However, the difference between the activation energies of wild-type RePC and R424S (in the absence of acetyl CoA) is approximately twice as large. The calculated values of the thermodynamic activation parameters at 30 °C show that acetyl CoA lowers the activation free energy (ΔG⧧) by ∼10 kJ mol−1, partially through an increase in activation entropy (less negative ΔS⧧) of ∼0.03 kJ mol−1 K−1 and partially through a decrease in the enthalpy of activation (ΔH⧧) of ∼6 kJ mol−1. Compared to that of wildtype RePC, R424S has a ΔG⧧ that is lower by 3.7 kJ mol−1 for the pyruvate carboxylation reaction in the absence of acetyl CoA that appears to be caused by a decrease in the activation enthalpy (ΔH⧧) of 13.1 kJ mol−1 that is offset to a degree by a decrease in activation entropy (more negative ΔS⧧).

■

DISCUSSION In the course of our studies of the structure and function of RePC, we have produced 54 mutants involving substitution of 24 different residues across all four domains of the enzyme. All of these mutant forms have been successfully expressed under conditions identical to those used in the work presented here, and the majority of these mutants have been studied and reported in published works.9,13−17,22,23 Thus, we have no reason to expect that that expression of mutants of Glu425 should require special expression conditions. Figure 2a shows that Glu425 not only interacts with Arg427 in the same subunit via its α-carbonyl oxygen but also interacts with Arg20 and Lys45 of an adjacent subunit that does not contain bound acetyl CoA, via its side chain carboxyl group. Glu425 is highly conserved across PCs from a wide range of organisms (Figure 2b), and it is quite likely that Glu425 forms essential intersubunit interactions that stabilize the tetrameric structure of the enzyme. Mutation of Glu425 is likely to perturb these

Figure 4. Modified Arrhenius plots depicting the temperature dependence of the catalytic rate constant for the pyruvate carboxylation reaction in the absence of acetyl CoA: wild-type RePC (■), R424S (●), and wild-type RePC in the presence of acetyl CoA (▲). The solid lines represent fits of the data using linear least-squares regression analysis. E

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

involved in direct interaction with acetyl CoA, R424S also showed 2-fold increase in k0cat relative to that of wild-type RePC, and in this respect, it is similar to the effects observed for D1018A and E1027A, which also do not interact directly with acetyl CoA,23 although in these cases the increases in k0cat were much larger. As mentioned above, the primary locus of action of acetyl CoA is in the biotin carboxylation reaction in the BC domain, with only relatively minor effects on the carboxyltransferase reaction in the CT domain to decarboxylate carboxybiotin and form oxaloacetate.27 This is demonstrated by the lack of an effect of acetyl CoA on the oxamate-induced decarboxylation of the oxaloacetate reaction that occurs completely in the CT domain (Table 3). This is mirrored by the relatively small effects of the mutations of Asp420, Arg424, and Arg429 on this reaction in the presence and absence of acetyl CoA (Table 3). Substitution of Arg424 and Arg429 with serine resulted in large increases in kcat for bicarbonate-dependent MgATP cleavage in the absence of acetyl CoA and smaller increases in its presence, relative to that of wild-type RePC. This was unlike mutations of Arg427 and Arg472 that resulted in large decreases in kcat in the presence and absence of acetyl CoA.22 However, mutation of Arg469 also resulted in increases in both k0cat and kcat.23 R424S exhibits the largest increases in k0cat and kcat for bicarbonate-dependent MgATP cleavage (Table 3). However, this is not reflected in the effects of the same mutation in the pyruvate carboxylation reaction, where the increase in k0cat is only 2-fold and kcat actually decreases. One possible explanation for this discrepancy in the effects of R424S on pyruvate carboxylation compared to MgATP cleavage is that the mutation has decreased the extent of coupling between the two reactions. However, the results of comparison of Pi release and oxaloacetate formation showed that there is essentially no difference in the coupling of the reactions between wild-type RePC and R424S in the presence or absence of acetyl CoA. Thus, R424S has a large effect on the rate-limiting step on the steady state bicarbonate-dependent MgATP cleavage reaction, which has been shown to be carboxybiotin decarboxylation at the CT domain.20,21 However, this reaction is performed in the absence of pyruvate, and this reaction step, in the pyruvate carboxylation reaction, is not likely to be so rate-limiting.21,28 The question of why the R424S and R429S mutations show greater positive effects on pyruvate carboxylation and on bicarbonate-dependent MgATP cleavage in the absence of acetyl CoA than in its presence arises. In their study of mutations of Arg469, Asp1018, and Glu1027, which showed similar types of effects, Choosangtong et al.23 focused on the differences in the interactions between these residues and other residues seen in the RePC tetrameric structure, depending on whether the residues are in the two subunits of RePC with acetyl CoA bound or in the other pair of subunits. As mentioned above, this asymmetry of the tetramer is not caused by acetyl CoA binding but is an inherent property of the RePC tetramer. Thus, the asymmetrical conformer appears to be a very stable form of the tetramer that thus crystallizes, with only two of the acetyl CoA binding sites occupied. As mentioned in the introductory section, the value of the Hill coefficient of >2 for the activation of RePC by acetyl CoA indicates that during catalysis in solution, all four allosteric binding sites may be occupied. Indeed, Yu et al.12 determined the crystal structure of SaPC to which four acetyl CoA molecules were bound, which was much more symmetrical than that of the RePC tetramer. Later, however, Lasso et al.7 reported that the SaPC tetramer

interactions with Arg20 and Lys45 and thus destabilize the quaternary structure of the enzyme, making it susceptible to proteolytic degradation. As shown in Figure 2a, Arg429 interacts directly with the 3′phosphate group of acetyl CoA, similar to Arg427, which has also previously been shown to interact with the 3′-phosphate,22 and Arg472, which interacts with the 5′-phosphates. In terms of the effect on the Ka of activation of the enzyme by acetyl CoA, the R429S mutation has a stronger effect (100-fold increase compared to that of wild-type RePC) than the R427S mutation (15-fold increase).22 This suggests the greater importance of Arg429 in acetyl CoA binding compared to Arg427, and indeed, Arg429 is a highly conserved residue in PCs from other organisms. However, from the effect of the R472S mutation on Ka (203-fold increase relative to that of wild-type RePC),22 Arg472 remains the most important residue in terms of acetyl CoA binding. Unlike the substitutions of Arg427 and Arg472 with lysine, which produced increases in Ka considerably larger than those caused by the serine mutations,22 the R429K mutation produced an increase in Ka much smaller than that caused by R429S. Adina-Zada et al.22 speculated that the reason for the apparently anomalous effects of the more conservative R427K and R472K mutations compared to the effects of the R427S and R472S mutations was that, while the substitute lysine residues could still interact with acetyl CoA, they lacked the ability to form interactions with surrounding residues (see Figure 2a), and this led to improper positioning of acetyl CoA in the binding pocket. This is clearly not the case for the R429K mutation, where the effect of the mutation on Ka is much attenuated compared to that of R429S. The reason for this is apparent in Figure 2a, which shows that when interacting with acetyl CoA, Arg429 does not have any interactions with other residues. Hence, in R429K, the lysine would still able to interact quite well with acetyl CoA to assist binding, but no interactions with other residues would have been disrupted by the substitution of lysine with arginine. Even though Asp420 and Arg424 form no direct interactions with bound acetyl CoA (Figure 2a), mutation of these residues did produce increases in Ka relative to that of wild-type RePC, with the effect in D420A being much smaller than that in R424S. The only interactions that the side chain groups of Asp420 and Arg424 form in the subunits containing bound acetyl CoA are with each other (Figure 2a). However, the αcarbonyl oxygen of Arg424 does interact with Arg427 (Figure 2a), and it is likely that effects of the mutation of Asp420 and Arg424 on acetyl CoA binding are mediated through this interaction, by changing the positioning of Arg427 relative to acetyl CoA. Substitution of Arg429 with serine, in addition to affecting acetyl CoA binding, also resulted in a reduction in the cooperativity of acetyl CoA activation, unlike R427S and R472S mutations that had little effect on the Hill coefficient.22 These results suggest that of the residues that directly interact with bound acetyl CoA (including Arg46923), Arg429 is the most important in determining the cooperativity of acetyl CoA action. All substitutions of arginine residues involved in acetyl CoA binding with serine resulted in decreases in kcat, relative to that of wild-type RePC, that varied between 35 and 70%.22,23 However, the substitution of Arg429 with serine resulted in a nearly 3-fold increase in k0cat relative to that of wild-type RePC, less than the 20-fold increase seen with R469S,23 but greater than that for R427S, which showed an only 60% increase, or R472S, which showed a 30% decrease.22 Despite not being F

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry does change between an asymmetrical and a symmetrical conformation that is dependent on the stage of the enzymecatalyzed reaction and that the half-of-the-sites activity alternates between pairs of subunits on opposite faces of the tetramer. The asymmetrical conformer is associated with the carboxylation of biotin in the BC domain, and the symmetrical conformer is associated with the decarboxylation of carboxybiotin in the CT domain.7 Choosangtong et al.23 postulated that the interactions of the residues in the asymmetrical structure of the RePC tetramer seen in their studies stabilized this structure and would thus constrain the interconversion between this conformer and the symmetrical conformer during the course of catalysis. As a consequence of this, mutations of residues that removed these interactions would allow the freer, more rapid interconversion of the two conformers and hence increase the rate of catalysis. Choosangtong et al.23 also postulated that one effect of acetyl CoA binding was to enhance this interconversion between the symmetrical and asymmetrical conformers of PC. This conformational interconversion would be required in the pyruvate carboxylation reaction and the steady state bicarbonate-dependent MgATP cleavage reaction, but not the oxamate-induced oxaloacetate decarboxylation reaction, which occurs completely at the CT domain. The effects of acetyl CoA on pyruvate carboxylation and MgATP cleavage and the lack of an effect of acetyl CoA on the oxamateinduced oxaloacetate decarboxylation reaction described in this work tend to support the proposal that acetyl CoA activation of PC occurs, at least in part, because of its effect on conformational interconversion. A similar argument also applies to the effects of the mutation of Arg424. The significant increases in the rate constants for oxamate-induced oxaloacetate decarboxylation seen in R429S suggest that while this mutation may affect the interconversion between asymmetrical and symmetrical conformers, there is an additional effect of this mutation that influences events at the CT domain. Examination of RePC structures shows that in subunits that do not contain bound acetyl CoA, Arg429 interacts with either the oxygen of the α-carbonyl group of Gln1037 (Figure 5a)9 or the carboxyl group of Glu1053 (Figure 5b).8 As can be seen in panels a and b of Figure 5, in the subunits with bound acetyl CoA, Arg429 is quite distant from Gln1037 and Glu1053. Thus, in the conversion of the asymmetrical RePC tetramer to the symmetrical conformer, these interactions will be broken, and the mutation of Arg429 to serine would probably result in the loss of these interactions and thus allow freer interconversion of the conformers in the absence of acetyl CoA. There is not an obvious explanation for why this mutation enhances oxamateinduced oxaloacetate decarboxylation. Because Gln1037 or Glu1053 is close to the BCCP domain, we can speculate that the R429S mutation has somehow facilitated the interaction of this domain (and its bound biotin) with the CT domain. Thus, the overall effect of the R429S mutation on the pyruvate carboxylation reaction may be attributed to a combination of effects on conformer interconversion and BCCP−biotin interaction with the CT domain. The explanation for the effects of the R424S mutation is more straightforward. In RePC structures, the guanidinium group of Arg424 maintains its interaction with Asp420 in subunits with and without bound acetyl CoA; however, in subunits that do not contain bound acetyl CoA, Arg424 can form a hydrogen bonding interaction with the carboxyl group of Glu439 (see Figure 6). The shortest distance between these

Figure 5. Positions Gln1037 and Glu1053 relative to Arg429 in the RePC subunits that contain bound acetyl CoA (colored residues, black labels) and subunits that do not contain the bound activator (gray residues and labels). (a) Residues from the structure of PDB entry 2QF7 of RePC. (b) Residues from the structure of PDB entry 3TW6 of RePC. Distances between Arg429 and the α-carbonyl oxygen of Gln1037 (a) and a carboxyl oxygen of Glu1053 (b) are indicated.

two residues is somewhat variable between subunits and structures with an average distance of 3.1 Å, while in the other set of subunits, the average distance is 4.1 Å. Again, the conversion from the asymmetrical conformer to the symmetrical conformer of RePC may involve disruption of this hydrogen bonding interaction. Again, substitution of Arg424 with serine would probably result in the loss of this interaction and facilitation of the interconversion between the RePC conformers and enhancement of the rate of pyruvate carboxylation in the absence of acetyl CoA. To obtain evidence to support the proposal that the R424S mutation results in the loss of a hydrogen bonding interaction with Glu439 leading to a larger k0cat for pyruvate carboxylation, thermodynamic activation parameters for the reaction at 30 °C were calculated using modified Arrhenius plots and the values of the measured rate constants at 30 °C. The Arrhenius plot (Figure 4) and activation energy (Table 4) reported in this work for wild-type RePC-catalyzed pyruvate carboxylation in the absence of acetyl CoA are not dissimilar to those described by Libor et al. for Bacillus stearothermophilus PC (BsPC) (56.3 kJ mol−1). However, unlike that for RePC in the presence of G

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry

greater activity of SaPC in the absence of acetyl CoA compared to that of RePC. It is possible that differences that are observed in the activation of PCs from different organisms by acetyl CoA are due to the variability in the interactions between residues in the allosteric domain and other residues that influence the conformational state of the PC tetramer and the ease of interconversion between conformers. In support of this proposition, we have seen that mutation of Asp420 did produce effects (if relatively small) on acetyl CoA activation of pyruvate carboxylation in RePC, but as shown in Figure 2b, this is not a residue that is at all conserved across PCs from different organisms. Similarly, although mutation of Arg424 did produce substantial effects on acetyl CoA activation of pyruvate carboxylation in RePC, it appears to be conserved only across the bacterial PCs considered and not the eukaryotic PCs (Figure 2b).

■

Figure 6. Positions of Asp420 and Glu439 relative to Arg424 in the RePC subunits that contain bound acetyl CoA (colored residues, black labels) and subunits that do not contain the bound activator (gray residues and labels). Figure based on the structure of PDB entry 3TW6 of RePC. Dashed lines indicate potential interactions between residues, and distances between Arg424 and Glu439 are indicated.

AUTHOR INFORMATION

Corresponding Authors

*Department of Biochemistry, Mahidol University, Bangkok, Thailand. Telephone: +662-201-5458. Fax: +61-8-354-7174. Email: [email protected]. *School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia. Telephone: +61-8-6488-3329. Fax: +61-8-6488-1148. E-mail: [email protected].

acetyl CoA, the Arrhenius plot for BsPC was biphasic, with a breakpoint at ∼30 °C. The activation energy for BsPC was 46.3 kJ mol−1 above 30 °C and 106.3 kJ mol−1 below 30 °C. If we examine the effect of the R424S mutation on the thermodynamic activation parameters of this reaction (Table 4), we can see that the decrease in ΔG⧧ compared to that of wild-type RePC was brought about by a reduction in ΔH⧧ of ∼13 kJ mol−1. This is well within the range of the bond enthalpies for a hydrogen bond and tends to support the mechanism proposed for the effect of the R424S mutation on pyruvate carboxylation and the more general proposal that some residues in the allosteric domain of RePC stabilize the asymmetric form of the tetramer by forming interactions with other residues, thus enhancing the activation energy required to attain the transition state in the conversion to the symmetrical conformer. Interestingly, the other effect of the R424S mutation was to increase the loss of entropy required to attain the transition state of the reaction, and this may arise from an increase in entropy of the asymmetrical conformer, i.e., a more disordered structure. In summary, we have completed the characterization of a series of arginine residues in the allosteric domain of RePC that are very important in the binding of acetyl CoA (Arg429). In addition, we have shown that another arginine residue (Arg424), despite having no direct contact with bound acetyl CoA, does influence binding of the activator. We have provided evidence that Arg424 forms an interaction with Glu439 in subunits without bound acetyl CoA that stabilizes the asymmetrical conformer of the RePC tetramer and constrains interconversion between the tetrameric conformers in the absence of acetyl CoA to inhibit pyruvate carboxylation. In SaPC, the residue equivalent to Glu439 is Ile432, and thus, the residue equivalent to Arg424 in SaPC (Arg417) does not form an interaction with another residue in the way it does in RePC. As noted by Choosangtong et al.,23 SaPC also lacks the interactions between Arg427 and Arg469 and other residues that are observed in RePC that were postulated to stabilize the asymmetrical conformer of that tetramer. Choosangtong et al.23 proposed that the lack of these interactions contributes to the

Funding

C.S. was supported by a Royal Golden Jubilee (RGJ-PhD) (PHD/0308/2551) scholarship co-funded between the Thailand Research Fund and Mahidol University. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS acetyl CoA, acetyl coenzyme A; PC, pyruvate carboxylase; RePC, R. etli pyruvate carboxylase; SaPC, S. aureus pyruvate carboxylase; BsPC, B. stearothermophilus pyruvate carboxylase; BC, biotin carboxylase; CT, carboxyl transferase; BCCP, biotin carboxyl carrier protein; PDB, Protein Data Bank.

■

REFERENCES

(1) 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. (2) Schar, 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. (3) MacDonald, M. J., Longacre, M. J., Langberg, E. C., Tibell, A., Kendrick, M. A., Fukao, T., and Ostenson, C. G. (2009) Decreased levels of metabolic enzymes in pancreatic islets of patients with type 2 diabetes. Diabetologia 52, 1087−1091. (4) Kumashiro, N., Beddow, S. A., Vatner, D. F., Majumdar, S. K., Cantley, J. L., Guebre-Egziabher, F., Fat, I., Guigni, B., Jurczak, M. J., Birkenfeld, A. L., Kahn, M., Perler, B. K., Puchowicz, M. A., Manchem, V. P., Bhanot, S., Still, C. D., Gerhard, G. S., Petersen, K. F., Cline, G. W., Shulman, G. I., and Samuel, V. T. (2013) Targeting pyruvate carboxylase reduces gluconeogenesis and adiposity and improves insulin resistance. Diabetes 62, 2183−2194. (5) 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.

H

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry (6) Sellers, K., Fox, M. P., Bousamra, M., 2nd, Slone, S. P., Higashi, R. M., Miller, D. M., Wang, Y., Yan, J., Yuneva, M. O., Deshpande, R., Lane, A. N., and Fan, T. W. (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. J. Clin. Invest. 125, 687−698. (7) Lasso, G., Yu, L. P., Gil, D., Lazaro, M., Tong, L., and Valle, M. (2014) Functional conformations for pyruvate carboxylase during catalysis explored by cryoelectron microscopy. Structure 22, 911−922. (8) 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. (9) Maurice, M., St, Reinhardt, L., Surinya, K. H., Attwood, P. V., Wallace, J. C., Cleland, W. W., and Rayment, I. (2007) Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme. Science 317, 1076−1079. (10) Sureka, K., Choi, P. H., Precit, M., Delince, M., Pensinger, D. A., Huynh, T. N., Jurado, A. R., Goo, Y. A., Sadilek, M., Iavarone, A. T., Sauer, J. D., Tong, L., and Woodward, J. J. (2014) The cyclic dinucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function. Cell 158, 1389−1401. (11) 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. (12) Yu, L. P., 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. (13) 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. (14) 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. (15) 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. (16) 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. (17) 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. (18) Wallace, J. C. (1985) Distribution and biological functions of pyruvate carboxylase in nature. In Pyruvate Carboxylase (Keech, D. B., and Wallace, J. C., Eds.) pp 5−64, CRC Press, Boca Raton, FL. (19) 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. (20) Branson, J. P., and Attwood, P. V. (2000) Effects of Mg(2+) on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase. Biochemistry 39, 7480−7491. (21) Legge, G. B., Branson, J. P., and Attwood, P. V. (1996) Effects of acetyl CoA on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase. Biochemistry 35, 3849−3856. (22) 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. (23) 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 Lett. 589, 2073−2079.

(24) Chapman-Smith, A., Turner, D. L., Cronan, J. E., Jr., Morris, T. W., and Wallace, J. C. (1994) Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase. Biochem. J. 302 (Part 3), 881−887. (25) Green, N. M. (1965) A Spectrophotometric Assay for Avidin and Biotin Based on Binding of Dyes by Avidin. Biochem. J. 94, 23C− 24C. (26) Sirithanakorn, C., Adina-Zada, A., Wallace, J. C., Jitrapakdee, S., and Attwood, P. V. (2014) Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate. Biochemistry 53, 7100−7106. (27) Attwood, P. V., and Wallace, J. C. (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. (28) 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. (29) Abd El Latif, A., El Bialy, B. E. S., Mahboub, H. D., and Abd Eldaim, M. A. (2014) Moringa oleifera leaf extract ameliorates alloxaninduced diabetes in rats by regeneration of beta cells and reduction of pyruvate carboxylase expression. Biochem. Cell Biol. 92, 413−419. (30) Attwood, P. V., Mayer, F., and Wallace, J. C. (1986) Avidin as a probe of the conformational changes induced in pyruvate carboxylase by acetyl-CoA and pyruvate. FEBS Lett. 203, 191−196. (31) 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.

I

DOI: 10.1021/acs.biochem.6b00548 Biochemistry XXXX, XXX, XXX−XXX