Plant Physiol. (1998) 116: 1443-1450

Partial Purification and Characterization of the Maize Mitochondrial Pyruvate Dehydrogenase Complex¹

Jay J. Thelen, Jan A. Miernyk, and Douglas D. Randall^*

Department of Biological Sciences (J.J.T.), and Department of Biochemistry (J.A.M., D.D.R.), University of Missouri, Columbia, Missouri 65211

	ABSTRACT

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The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1-, E1-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg²⁺. Michaelis constants for the substrates were 47, 3, 77, and 1 µm for pyruvate, coenzyme A (CoA), NAD⁺, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD⁺ and CoA, and the inhibition constants were 15 and 47 µm, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg²⁺.

	INTRODUCTION

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The PDC catalyzes the oxidative decarboxylation of pyruvate to form acetyl-CoA and NADH. The PDC is composed of three fundamental enzymatic components: PDH (E1, EC 1.2.4.1), dihydrolipoyl transacetylase (E2, EC 2.3.1.12), and dihydrolipoamide dehydrogenase (E3, EC 1.8.1.4). The PDC from mammals (Gopalakrishnan et al., 1989), yeast (Behal et al., 1989), and perhaps plants (Taylor et al., 1992) contains an associated E3-binding protein. mtPDCs also contain two regulatory enzymes, PDH kinase and P-PDH phosphatase, which regulate PDC by reversible phosphorylation of the -subunit of PDH (E1). Mammalian and yeast mtPDC have a central pentagonal dodecahedryl core of E2-subunits to which the E1- and E3-subunits attach (Patel and Roche, 1990; Stoops et al., 1997). This E2 core comprises 20 trimers of a single polypeptide (Patel and Roche, 1990). Six to 12 E3 dimers, 6 to 12 E3-binding protein monomers, and 20 to 30 E1-₂₂ heterotetramers bind noncovalently to the E2 core (Patel and Roche, 1990).

The metabolic location of mtPDC and the irreversible nature of the reaction suggest that it is a site for regulation of mitochondrial carbon metabolism (Randall et al., 1996). All PDCs studied thus far are regulated by product inhibition (Patel and Roche, 1990; Luethy et al., 1996). In higher eukaryotes mtPDC activity is also regulated by reversible phosphorylation catalyzed by a PDH-specific protein kinase and a P-PDH-specific phosphatase (for review, see Patel and Roche, 1990; Randall et al., 1996).

The importance of mtPDC in controlling primary carbon metabolism is reflected by the many literature reports. However, there are a limited number of reports describing research on plant mtPDCs (for review, see Randall et al., 1996). Furthermore, our understanding of the regulation of plant mtPDC is derived from a limited number of C₃ species (e.g. pea, broccoli [Rubin and Randall, 1977], and castor bean [Rapp et al., 1987]).

In most C₃ species, leaf mtPDC is reversibly inactivated in the light in a photosynthesis- and photorespiration-dependent manner (Budde and Randall, 1990; Gemel and Randall, 1992). This is most likely the result of photorespiratory Gly metabolism that occurs in the leaf mitochondria during photosynthesis. Gly oxidation generates large amounts of NADH to support the necessary mitochondrial ATP production and NH₄⁺ to stimulate PDH kinase (Schuller et al., 1993). Consequently, mtPDC is negatively regulated as Gly oxidation increases, and all indications are that this light inactivation is caused by reversible phosphorylation of mtPDC. Pyruvate is the most effective inhibitor of mtPDC phosphorylation/inactivation (Schuller and Randall, 1990). In many C₄ species such as maize (Zea mays L.), pyruvate is a major metabolite in the photosynthetic CO₂-fixation process, and C₄ species lack significant photorespiration (Hatch, 1987). Therefore, light-dependent inactivation of mtPDC would not be expected. However, light-dependent inactivation of mtPDC was observed in maize leaves (Gemel and Randall, 1992), suggesting that the regulation of mtPDC may be different in maize and other C₄ plants.

To establish the properties and regulation of mtPDC in maize as a representative C₄ plant, we have undertaken a thorough examination of maize mtPDC beginning with nonphotosynthetic tissue to establish a baseline before proceeding to the characterization of the leaf mtPDCs, which will involve the two different cell types involved in C₄ photosynthesis. This report describes the partial purification and characterization of mtPDC from etiolated shoots of maize.

	MATERIALS AND METHODS

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Maize (Zea mays B73) seeds were obtained from the Illinois Seed Foundation (Urbana). Mitochondria were isolated from etiolated shoots as previously described (Hayes et al., 1991). Protein was quantified according to the method of Bradford (1976) using BSA as the standard. All other materials were from Sigma or Fisher Scientific.

Activity Assays

Electrophoresis and Immunoblot Analysis

Purification of Mitochondrial PDC

	RESULTS

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Plants are unique in that they contain a plastid PDC isoform (Williams and Randall, 1979; Camp and Randall, 1985) in addition to mtPDC. Therefore, to study the mitochondrial isoform, it was necessary to demonstrate that the purified mitochondria had low plastid contamination. Using TPI as the plastid marker enzyme, it was established that only 0.05% of the total TPI activity was present with the purified mitochondria.

Starting with 955 g fresh weight of etiolated maize shoots, 0.4 mg of highly enriched PDC was obtained, corresponding to 1.4% of the total mitochondrial protein (Table I). Almost 30% of total PDC activity was recovered, with a 21-fold enrichment. The specific activity of the partially purified maize mitochondrial PDC (0.81 µmol min¹ mg¹) was lower than values previously reported for purified cauliflower mtPDC (5.4 µmol min¹ mg¹, enriched approximately 100-fold; Randall et al., 1977) and purified broccoli mtPDC (6.3 µmol min¹ mg¹, enriched approximately 200-fold; Rubin and Randall, 1977). The peak of PDC activity consistently sedimented at 30% glycerol (Fig. 1A), which is similar to the peaks of other mtPDCs but larger than the peak of either plastid or Escherichia coli PDC (Camp and Randall, 1985). Compared with the sedimentation profiles of other mtPDCs, the molecular mass of the maize mtPDC was estimated at about 8000 to 9000 kD (Patel and Roche, 1990).

View larger version (55K): [in this window] [in a new window]   Figure 1. A, Fractionation profile for a typical rate-zonal glycerol gradient. Approximately 3 mg of 400K enzyme was loaded onto this gradient. Fractions 19 through 30 were pooled and concentrated for the SDS gel shown in Figure 2. B, Coomassie blue-stained SDS-PAGE of odd-numbered glycerol-gradient fractions 7 to 33. Positions of protein standards are indicated on the left (in kilodaltons). The position of the 110-kD protein is indicated on the right with an arrowhead. U, Units.

PDC Subunit Composition

View larger version (53K): [in this window] [in a new window]   Figure 2. SDS-PAGE and corresponding immunoblots of lysed mitochondria (total mito), the supernatant from the 100,000g centrifugation (100K super), the 400K enzyme (400K pellet), and the pooled mtPDC activity fraction from the glycerol-gradient fractionation (glycerol). A, Coomassie blue-stained SDS-PAGE gel loaded with 5 µg of protein per lane. Positions of protein standards are indicated on the left and calculated molecular mass values of the predominant bands in the glycerol fraction are indicated on the right (in kilodaltons). B, Four replica protein blots of the fractions in A were probed with anti-subunit antibodies. One microgram of protein was loaded per lane. The molecular masses of the protein bands are indicated on the left (in kilodaltons).

Monoclonal antibodies to maize E1 recognized the 43-kD band in immunoblots (Fig. 2B). However, this single 43-kD band presented six isoelectric forms on immunoblots after two-dimensional IEF/SDS-PAGE separation (Fig. 3B). These multiple isoelectric forms may reflect the phosphorylation of multiple residues creating a gradient of phosphoproteins. Polyclonal antibodies to recombinant Arabidopsis E1 recognized a 40-kD polypeptide (Fig. 2B) highly enriched in the pooled glycerol-gradient fraction. In light of the strong evidence that PDC and GDC in pea mitochondria share identical E3 components (Bourguignon et al., 1996), antibodies raised against the pea L-protein of GDC were used to probe maize mtPDC. These antibodies recognized a 62- to 63-kD doublet that was enriched in the glycerol-gradient-purified fraction (Fig. 2B). Antibodies that recognize the 76-kD pea E2-subunit reacted with a 52- to 53-kD maize doublet that was enriched throughout the PDC purification (Fig. 2B).

View larger version (29K): [in this window] [in a new window]   Figure 3. Two-dimensional gel electrophoresis of glycerol-gradient-enriched maize mtPDC and corresponding immunoblots. A, Ten micrograms of glycerol-gradient-enriched mtPDC was resolved by IEF in the first dimension followed by SDS-PAGE. B and C, One microgram of protein was resolved as in A, transferred to nitrocellulose, and probed with antibodies (Ab) to the E1- and E1-subunits. The molecular masses of the protein bands are indicated on the left (in kilodaltons).

The identity of the 52- to 53-kD doublet was further established by microsequencing the N terminus of these two proteins. The N-terminal amino acid sequence of the 52-kD protein (Fig. 4) had the highest similarity to a mammalian dihydrolipoamide transacetylase (E2), according to BLAST (Altschul et al., 1990), an amino acid alignment algorithm. N-terminal sequencing of the 53-kD protein revealed that it is related to the 52-kD protein (Fig. 4). Aligning the N-terminal sequence of the maize 52-kD protein with yeast (Niu et al., 1988), Arabidopsis (Guan et al., 1995), and human (Coppel et al., 1988) deduced E2 amino acid sequences showed the highest similarity within the lipoyl domains.

View larger version (26K): [in this window] [in a new window]   Figure 4. Amino acid alignment of N-terminal amino acid sequences for maize 52- and 53-kD proteins and deduced amino acid sequences for yeast, Arabidopsis, and human E2-subunits. The number of amino acid residues (aa) before the homologous region is indicated to the left of the sequences. Shading indicates an identical amino acid. X indicates a cycle of Edman degradation for which no determination was made.

Reaction Requirements and Kinetic Properties

Maize mtPDC required CoA, NAD⁺, thiamine pyrophosphate, and divalent cations for activity and did not use NADP⁺. The 400K enzyme specifically decarboxylated pyruvate and exhibited only minor activity with 2-oxobutyrate (15%), 3-hydroxypyruvate (6%), and 3-hydroxybutyrate (5%). No activity was seen with the branched-chain keto acids 2-oxoisovalerate and 2-oxoisocaproate, or with 2-oxoglutarate. The dialyzed maize 400K enzyme had an absolute requirement for divalent cations (Fig. 5), with Mg²⁺, Mn²⁺, and Ca²⁺ all able to restore activity.

View larger version (18K): [in this window] [in a new window]   Figure 5. Divalent cation requirement for the 400K enzyme. The 400K enzyme was dialyzed for 2 h in 2 L of 2 mm EDTA and 2 mm EGTA to remove endogenous cations, and then dialyzed twice in 2 L of 20 mm Tes, pH 7.5, and 0.5 mm DTT to remove the chelators. Enzyme was added to an assay vessel that contained divalent cations and necessary components. Rates are expressed as relative percentages of the maximum rate (0.093 µmol min1 mg1).

K_m values were determined under optimal conditions of pH and saturating concentrations of nonvariable substrates. Most of the K_m values for maize mtPDC were in the range of those reported for other plant PDCs (Table II). The exception was TPP; its K_m value was 10-fold higher than that of pea. This may explain why the maize complex, unlike the pea complex (Miernyk and Randall, 1987), required exogenous TPP for activity. The K_i for NADH was 5-fold lower than the K_m for NAD⁺, whereas the K_i for acetyl-CoA was much higher than the K_m for CoA, suggesting that NADH could be a more potent product inhibitor (Table II). Both products were competitive inhibitors with respect to their substrates (data not shown).

Intermediates of the Krebs cycle, amino acids, and polyamines had little effect on the activity of the 400K enzyme when tested at 2 mm. Hydroxypyruvate, previously shown to be a noncompetitive inhibitor of PDC (Randall et al., 1977), inhibited maize PDC by 36%. Ali et al. (1993) reported that the E1-subunit of mammalian PDC contained an essential Cys (Cys-62 in humans). Mutation of this Cys to Ala or derivatization by sulfhydryl reagents completely inactivated the mammalian enzyme. Because this essential Cys is not present in prokaryotic or plastid (Johnston et al., 1997) forms of PDC, we determined the effect of sulfhydryl reagents on maize mtPDC. Mersalyl, p-hydroxymercuribenzoate, and N-ethylmaleimide rapidly inactivated the maize mtPDC.

ATP-Dependent Inactivation

View larger version (15K): [in this window] [in a new window]   Figure 6. A, ATP-dependent inactivation of the 400K enzyme. Equimolar amounts of Mg2+ and ATP were added to PDC preparations to the final concentrations indicated. The control did not have any MgATP added. One-hundred-microgram samples of enzyme were removed at various time intervals and assayed for activity. Activity is expressed as a percentage of the control (0.16 µmol NADH formed min1 mg1 protein) at time 0. B, The effect of Mg2+ on reactivation of P-PDC. The 400K enzyme was incubated with 200 µm MgATP until the inactivation of mtPDC ceased. Free ATP was then removed with 2.5 units of hexokinase (HK) and 2 mm Glc at 30 min. The 400K enzyme was then divided into four aliquots to which EDTA, MgCl2, or buffer (control) was added to the final concentrations indicated.

	DISCUSSION

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Rate-zonal density-gradient centrifugation of maize mitochondrial matrix protein yielded a distinct peak of PDC activity with a specific activity ranging from 1.2 to 1.7 units mg¹ protein (Fig. 1A), and the predominant polypeptides in this peak were identified as the E1-, E1-, E2-, and E3-subunits. Additionally, a 110-kD Coomassie blue-stained polypeptide was observed; however, this protein did not peak with PDC peak activity (Fig. 1B) and was not recognized by any PDC antibodies. Polypeptides of 50 and 55 kD were also observed in heavily loaded lanes; however, the 55-kD polypeptide also did not peak with PDC peak activity. The unidentified 50-kD protein was probably not PDH kinase or P-PDH phosphatase, since glycerol-gradient-enriched mtPDC lacked these activities. Alternatively, it could have been an E3-binding protein homolog, which is also a 50-kD protein in yeast (Behal et al., 1989).

A comparison of the apparent and calculated sizes of plant mtPDC subunits shows that only the maize E1-subunit is identical in size to other plant E1-subunits, whereas the E1- and E3-subunits are 2 to 3 kD larger (Table III). In contrast, the maize E2-subunit (52 kD) is much smaller than other plant E2-subunits (76 kD), which can be explained by its variable multidomain structure.

The E2-subunit possesses a multidomain structure, with a lipoyl domain(s) connected by flexible linkers to the E1-/E3-binding domains followed by the catalytic domain (Reed and Hackert, 1990; Perham, 1991). The flexible lipoyl domains allow active-site coupling between the E1- and E3-subunits for the following series of reactions. The E1 reductively acylates the covalently bound lipoate within the E2 lipoyl domain. The E2-subunit catalyzes the acyl-transfer step to CoA, and E3 catalyzes the reoxidation of the dihydrolipoyl moiety using NAD⁺ as the electron acceptor. In addition to having a catalytic role, the inner (second) lipoyl domain of mammalian E2 binds the kinase and phosphatase regulatory components (Liu et al., 1995; Chen et al., 1996).

The number of E2 lipoyl domains found in nature is variable (Reed and Hackert, 1990). Multiple, tandemly repeated lipoyl domains have been observed in the E2-subunits described previously for all organisms except bacilli and yeast (Perham, 1991). Although the tandemly repeated lipoyl domains are functional (Allen et al., 1989), only one is required for E2 catalytic or complex function (Guest et al., 1985; Machado et al., 1992). The considerably smaller size of the maize E2 can be explained if only one lipoyl domain is present. A single lipoyl domain may be attributable to either novel gene structure or proteolysis. Proteolysis is unlikely, since we observed only the 52- to 53-kD band for E2 with purified mitochondria lysed directly into SDS-PAGE sample buffer followed by boiling (data not shown).

Support for the presence of a single lipoyl domain is found in the reduced size of the maize E2, the N-terminal amino acid sequence (which is most similar to the single E2 lipoyl domain from yeast), and its similarity to the inner lipoyl domain of Arabidopsis and human E2. The properties of the maize E2 are consistent with previous findings, i.e. a single lipoyl domain is sufficient for complex activity and appears to be sufficient for binding the kinase and phosphatase, although maybe not as tightly as with the mammalian complex, since both the kinase and the phosphatase can be stripped away during the glycerol-gradient purification. Molecular analysis shows that the E2 from Arabidopsis has multiple lipoyl domains (Guan et al., 1995), so it will be interesting to determine if this is true for other plant species or if single lipoyl domains are characteristic of maize alone.

The pyruvate and CoA K_ms for maize mtPDC are similar to those from pea (Miernyk and Randall 1987), but unlike the pea mtPDC the maize complex has a lower K_m for NAD⁺ and a higher K_m for MgTPP, which may reflect differences in the E3 and E1 components. The high K_i for acetyl-CoA in relation to other plant PDCs may reflect a different in vivo environment for the maize mtPDC or the various functions of the maize mitochondria.

The maize mtPDC requires divalent cations for catalytic activity. Of the divalent cations tested, Mg²⁺ best satisfied this requirement, as determined by the V_max/K_m ratio for the three cations Mg²⁺ (2.3), Mn²⁺ (1.3), and Ca²⁺ (1.0). The K_m for Mg²⁺ was approximately 40 µm, considerably lower than that of pea mtPDC (360 µm; Miernyk and Randall, 1987) and pea plastid PDC (1 mm; Camp and Randall, 1985), suggesting that the divalent cation requirement does not have regulatory significance in maize. All plant PDCs except mtPDC from cauliflower (Randall et al., 1977) will accept Mn²⁺ and Ca²⁺ as Mg²⁺ substitutes.

The maize mtPDC is capable of regulation by reversible phosphorylation. Increasing amounts of MgATP completely inactivated mtPDC, although reactivation immediately ensued, indicating that PDC activity reflects the relative activities of the regulatory kinase and phosphatase. MgATP concentrations below saturation will not entirely inactivate mtPDC, even after extended periods. This can be explained by contaminating ATPase activity, high P-PDH phosphatase activity, multiple phosphorylation sites that coordinate full inactivation, or all of the above.

In summary, we have partially purified PDC from maize mitochondria and identified the catalytic subunits by immunoblot analysis. The molecular masses of the maize PDC subunits are similar to those of other plant PDCs, the exception being the E2-subunit, which was 23 kD smaller than pea E2. Overall, the kinetic properties of maize mtPDC were similar to those of other plant mtPDCs, although slight differences were observed with regard to the divalent cation and TPP requirement, as well as the product inhibitor acetyl-CoA. The degree of similarity between maize mtPDC and C₃-plant mtPDCs was somewhat surprising considering the differences in pyruvate metabolism.

	FOOTNOTES

   Received September 24, 1997; accepted December 23, 1997.

	ABBREVIATIONS

Abbreviations: 400K enzyme, the pellet from the 400,000g centrifugation. GDC, Gly decarboxylase complex. mtPDC, mitochondrial pyruvate dehydrogenase complex. PDC, pyruvate dehydrogenase complex. PDH, pyruvate dehydrogenase. P-PDH, phosphopyruvate dehydrogenase. TPI, triose phosphate isomerase. TPP, thiamine pyrophosphate.

	ACKNOWLEDGMENTS

The authors are grateful for discussions and critical reading by Dr. Michael H. Luethy. We also thank Professor Thomas E. Elthon and Dr. Gautum Sarath for the protein microsequencing performed at the Protein Core Facility, University of Nebraska-Lincoln.

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