©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutagenesis Studies of the Phosphorylation Sites of Recombinant Human Pyruvate Dehydrogenase
SITE-SPECIFIC REGULATION (*)

Lioubov G. Korotchkina , Mulchand S. Patel (§)

From the (1)Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammalian pyruvate dehydrogenase () (E) is regulated by phosphorylation-dephosphorylation, catalyzed by the E-kinase and the phospho-E-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E, several human E mutants were made with single, double, and triple mutations by changing Ser to Ala. Mutation at site 1 but not at sites 2 and/or 3 decreased E specific activity and also increased K values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E. However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E-phosphatase resulting in complete reactivation of the E. The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.


INTRODUCTION

Pyruvate dehydrogenase (E)()is the first component of the pyruvate dehydrogenase complex (PDC). E catalyzes the oxidative decarboxylation of pyruvic acid and reductive acetylation of lipoic acid residues of dihydrolipoamide acetyltransferase (E). E transfers acetyl moieties to CoA, and lipoic residues are reoxidized by dihydrolipoamide dehydrogenase (E) with the formation of NADH. Mammalian E is a tetramer () with molecular mass of 154 kDa(1, 2) . It has two active sites(3) , that are most probably composed of amino acid residues from both E and E subunits(4, 5) . Several active site residues (cysteine, tryptophan, arginine, lysine, and histidine) have been found to take part in active site formation(6, 7) . Three of these amino acid residues were identified in bovine E: namely, the Cys-62 equivalent of human -subunit(8) , Trp-135 of the human -subunit,()and Arg-239 of the human -subunit.()E is regulated in the PDC by phosphorylation-dephosphorylation carried out by specific regulatory enzymes: E-kinase and phospho-E-phosphatase, respectively(9, 10) . Three serine residues were shown to be phosphorylated and designated site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203) because of the extent and the rate of phosphorylation(11) . Phosphorylation of site 1 was correlated with major inactivation of the enzyme; and half-of-the-site reactivity was exhibited as phosphorylation of only one serine residue of one of the two -subunits was sufficient for complete inactivation of the enzyme(11) . The roles of sites 2 and 3 remain to be determined. The mechanism of inactivation is also not well understood. It was found that phosphorylation of bovine E inhibited all partial reactions leading to formation of 2-hydroxyethylidene-thiamin pyrophosphate (HETPP)(12) . Spectral studies on pigeon breast muscle E showed that phosphorylated E was able to bind thiamin pyrophosphate (TPP) (with less affinity) and was able to interact with HETPP, but could not bind the substrate pyruvate(13) .

We recently coexpressed the mature forms of human E and E subunits in Escherichia coli M15 cells, and recombinant human E was purified using affinity chromatography (5). This coexpression system has permitted a study of the regulation of E by phosphorylation through the use of site-directed mutagenesis. This paper describes a study of mutants of the three phosphorylation sites. All three sites were found to be phosphorylated by the E-kinase bound to the E, independent of the other two sites with concomitant inactivation of the enzyme and were dephosphorylated by the phospho-E-phosphatase with resulting reactivation of E. Replacement of serine by alanine at site 1 resulted in a reduction in specific activity of native E.


EXPERIMENTAL PROCEDURES

Materials

Restriction and modification enzymes were purchased from Boehringer Mannheim. Ni-nitrilotriacetic-agarose (Ni-NTA-agarose) was obtained from Qiagen. pQE-9 and E. coli M15 cells were kindly provided to us by Dr. Stuart F. J. Le Grice of Case Western Reserve University, and pQE-6HE1, pQE-3-E, and pQE-9-6HE/E were recently constructed in this laboratory(5) . Recombinant human dihydrolipoamide dehydrogenase (E) was expressed using a pQE-9-6HE3 expression vector in E. coli M15 and purified in this laboratory.()Bovine kidney E, the dihydrolipoamide acetyltransferase-protein X-E kinase (E-X-kinase) subcomplex, and the phospho-E-phosphatase were a generous gift of Dr. Thomas E. Roche of Kansas State University. Recombinant E-kinase was kindly provided by Dr. Robert Harris of Indiana University. Bovine heart E-X-kinase was purified by a modified method (14). [-P]ATP (25-50 Ci/mmol) was purchased from ICN.

Site-directed Mutagenesis

Site-directed mutagenesis was carried out by polymerase chain reaction (PCR). The mutations in the E cDNA of the site 1-MS1 (S264A), site 2-MS2 (S271A), or site 3-MS3 (S203A) were made by a two-stage procedure (15) using pQE-9-6HE, single mutagenic primer (MS1, MS2, or MS3) and two primers for 5` and 3` ends including BamHI sites preceding the first codon and after the stop codon of E sequence. The amplified, mutated E cDNA was digested with BamHI and ligated to pQE-9 (enabling polyhistidine extension at the NH terminus of the recombinant protein) yielding pQE-9-6HE-MS1 (MS2 or MS3). For construction of a coexpression vector pQE-9-6HE-MS1/E, pQE-9-6HE-MS1 was digested with SalI and PvuI to remove the promoter region and E cDNA, and ligated with fragment of pQE-3-E digested with XhoI and PvuI, having the E sequence (Fig. 1). In the same way pQE-9-6HE-MS2/E1 and pQE-9-6HE-MS3/E were constructed. For construction of pQE-9-6HE-MS1,2/E1 (with mutations at sites 1 and 2), pQE-9-6HE-MS1 was used as a template for PCR with a mutagenic primer-MS2, and a coexpression vector was constructed as described above. For construction of pQE-9-6H-E-MS1,3/E (with mutations at sites 1 and 3), pQE-9-6HE-MS1/E and pQE-9-6HE1-MS3/E1 were digested with XhoI and BglII and fragments carrying the mutations were ligated. In the same way pQE-9-6HE-MS2/E and pQE-9-6H-E-MS3/E1 were combined to obtain pQE-9-6H-E-MS2,3/E (with mutations at sites 2 and 3), and pQE-9-6H-E-MS1,2/E and pQE-9-6H-E-MS3/E were used for construction of pQE-9-6H-E-MS1,2,3/E. The vectors for expression of the E mutants were transformed into E. coli M15 cells containing the pDMI.1 plasmid, producing the lac repressor. The correct orientation and the sequences of each human E cDNA in the constructed vectors were confirmed by restriction enzyme digestion analyses and sequencing(16) .


Figure 1: Construction of a pQE-9-6HE1-MS1/E1 expression vector.



Overexpression and Purification of Recombinant Wild-type and Mutant Es

Overexpression and purification of recombinant wild-type and mutant Es using affinity chromatography on Ni-nitrilotriacetic-agarose (Ni-NTA-agarose) column were performed as described recently(5) . Ten mg of recombinant Es were obtained from 4 liters of bacterial culture.

Measurements of Activity and Kinetic Parameters

Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard(17) . Activity of E was determined in three assay systems: (i) by reduction of NAD after reconstitution with purified bovine E-X-kinase subcomplex and human recombinant E according to the method of Roche and Reed (18); (ii) by the rate of reduction of the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) at 600 nm(19) ; and (iii) by the production of CO from [1-C]pyruvate(20) . The last assay was performed in the presence and absence of KFe(CN) to study oxidative and nonoxidative decarboxylation of pyruvate, respectively. One milliunit of enzyme activity is defined as 1 nmol of substrate used or product formed per min at 37 °C. K values for TPP and pyruvic acid were determined by the reconstitution assay and assay with DCPIP by varying the concentration of TPP from 0.05 to 200 µM and the pyruvic acid concentration from 1 to 300 µM. Linear parts of the time curves were used for determination of kinetic parameters using double recipropcal plots.

Phosphorylation and Dephosphorylation of Recombinant Proteins

Phosphorylation of wild-type and mutant Es was performed as described previously(21) . To measure activity changes, nonradioactive ATP was used during incubation and aliquots were withdrawn at specified times from the reaction mixture to a 1-ml cuvette to measure activity using the reconstitution assay. To determine incorporation of P from [-P]ATP into protein, E-X-kinase subcomplex was first incubated with nonradioactive ATP (5-150 µM) for 5 min (to exclude phosphorylation of a trace amount of E bound to E), followed by the addition of [-P]ATP (5-150 µM, 1.25-13.0 µCi) and recombinant E or E mutants. In the experiments with purified recombinant E-kinase, preincubation was excluded and reaction was started by the addition of [-P]ATP (20-200 µM) (12.5 µCi). The aliquots taken at different time intervals were applied to dry paper discs presoaked in trichloroacetic acid and the samples were processed as described previously(21) . P incorporation was determined by scintillation counting (21) and expressed as mole of P incorporated per mole of tetrameric E. The rates of incorporation (mol P/mol Emin) were determined from the initial slopes of the time courses of P incorporation. k of inactivation (min) were calculated from the semilog plots of inactivation during phosphorylation. To demonstrate the incorporation of radioactivity in the subunit, E proteins were separated by SDS-PAGE and dry gels were autoradiographed.

To study dephosphorylation, recombinant E proteins were phosphorylated by the E-X-kinase from bovine kidney in the presence of 50-200 µM [-P]ATP for 45-100 min, and the phosphorylation was stopped by the addition of 8 mM glucose and 0.5 units of hexokinase to scavenge ATP. To study P release Es were extensively washed on Microcon filters to remove labeled glucose 6-phosphate. This step was excluded during reactivation experiments as it gave the same results. Dephosphorylation was performed in the presence of 10 mM MgCl, 100 µM CaCl, and 1 µg of phospho-E-phosphatase per 10 µg of phosphorylated E at 30 °C for up to 60 min. Aliquots taken at different time intervals were analyzed for both activity and P release in soluble fraction after protein precipitation with trichloroacetic acid. k of dephosphorylation and reactivation were calculated from semilog plots of the time courses of P release and increase of the specific activity.


RESULTS

Activity Determination of E Mutants

To examine the role of the three phosphorylation sites, seven different mutants were made by site-directed mutagenesis. Ser-264, Ser-271, Ser-203, corresponding to phosphorylation sites 1, 2, and 3 in the E sequence, were changed to alanine, either individually or in combination as: MS1 (E with mutated site 1), MS2, MS3, MS2,3, MS1,3, MS1,2, and MS1,2,3. Wild-type E and E mutants were individually overexpressed in E. coli and purified using Ni-nitrilotriacetic-agarose chromatography as described previously(5) . Approximately 10 mg of each enzyme were obtained from 4 liters of bacterial culture with a purity of about 94% based on densitometry of the SDS-PAGE gel.

The activities of these enzymes were measured using three different assays: (i) a reconstitution assay, (ii) a DCPIP assay, and (iii) a CO assay in the presence and absence of KFeCN as an electron acceptor. The results of the reconstitution assays are shown in Fig. 2. Similar results were observed using the other two assays (results not shown). Each mutant exhibited activity in all three assays. There was little decrease in the specific activity of the E phosphorylation sites 2 and/or 3 mutants, but the E specific activity was reduced by 50-70% (relative to wild-type E) for all mutants having an alteration at site 1.


Figure 2: Activity of the E mutants determined by the reconstitution assay. Values are the means ± S.E. of two to five measurements for two preparations of each enzyme. 100% corresponds to 14.9 units/mg.



shows the K values for TPP and pyruvic acid measured separately by the reconstitution assay and DCPIP assay. In each site 1 mutant (MS1, MS1,3, MS1,2, and MS1,2,3) a substantial increase in K values for both pyruvate and TPP was observed which is not seen in site 2 or 3 mutants (E, MS2, MS3, and MS2,3).

Phosphorylation of E Mutants by the E Kinase in the Presence of E

Fig. 3shows the time dependent inactivation of the wild-type and mutant Es during incubation with ATP and the E-X-kinase subcomplex (see ``Experimental Procedures''). Based on the results, the Es can be divided into 4 groups: Group 1, having site 1 intact (wild-type E, MS2, MS3, and MS2, 3); Group 2, with mutation of site 1, but intact site 2 (MS1 and MS1, 3); Group 3, with mutation of sites 1 and 2, but site 3 intact (MS1, 2); Group 4, with mutations of all three phosphorylation sites (MS1, 2, 3). Native E, MS2, MS3, and MS2,3 (Group 1) behaved similarly to one another during inactivation at ATP concentrations of 1-10 µM (Fig. 3A). Complete inactivation was achieved after 10 min with 10 µM ATP. However, the inactivation of the MS1 and MS1,3 (Group 2) was complete only after 30 min incubation with 40 µM ATP (Fig. 3B), and the MS1,2 (Group 3) needed higher ATP concentrations, up to 400 µM, and more than 60 min for complete inactivation (Fig. 3C). Thus, all Es were inactivated by E-X-kinase-dependent phosphorylation. However, the range of ATP concentration necessary for complete inactivation depended upon the mutant used, reflecting site-specificity. Mutants of Groups 2 and 3 achieved inactivation by phosphorylation at higher ATP concentration than mutants of Group 1.


Figure 3: Time course of E inactivation during phosphorylation by the E-X-kinase subcomplex. A, inactivation of Group 1 enzymes; B, inactivation of Group 2 enzymes; and C, inactivation of Group 3 enzyme. Ten µg of each E were incubated at 30 °C with 8 µg of the E-X-kinase and various ATP concentrations: 0 µM (*); 1.0 µM (); 1.6 µM (); 2.5 µM (); 4.0 µM ( ); 10 µM (); 25 µM (); 40 µM (); 100 µM (┌); and 400 µM (). Aliquots (1 µg of E) were taken to measure activity by the reconstitution assay.



To compare the regulatory behavior of different mutants the inactivation and the corresponding incorporation of P were determined at a fixed ATP concentration (40 µM) (Fig. 4). The level of incorporation (Fig. 4B) depended upon the number of sites available and duration of incubation. E with all the three potential phosphorylation sites available showed the maximum level of incorporation (2.7 mol of P/mol of E); MS3, MS2, and MS1 with only 2 sites available showed less phosphorylation (1.1-1.7 mol of P/mol of E) and minimum incorporation (0.4-0.6 mol of P/mol of E) corresponded to phosphorylation of individual sites 1 (MS2,3), 2 (MS1,3), and 3 (MS1,2). The maximal level of incorporation achieved at 270 µM ATP was: 3.0 for E; 1.5-1.8 for mutants with two sites; and 0.5-1.0 for mutants with only one phosphorylation site available (results not shown). There was no incorporation of P into the MS1,2,3 mutant and as expected there was no inactivation of this enzyme (Fig. 4). For this reason MS1,2,3 was used as a control in all the experiments concerning phosphorylation. Data presented in Fig. 4(B and C) showed that all three phosphorylation sites can incorporate P during incubation with the E-X-kinase subcomplex and [-P]ATP.


Figure 4: A, inactivation of Es during phosphorylation by the E-X-kinase subcomplex in the presence of 40 µM ATP. Activity was measured in the reconstitution assay. 100% activity of each curve corresponded to the activity of each E at zero time. B, P incorporation from [-P]ATP (40 µM) into Es by the E-X-kinase subcomplex. Ten µg of the E and 8 µg of the E-X-kinase were used in each reaction. Incorporation was measured by counting paper discs in a scintillation counter. Phosphorylation in control tubes containing only the E-X-kinase (without E) was subtracted. C, autoradiography of the E after incorporation of -P followed by separation of proteins in SDS-PAGE.



The difference in the rate of inactivation of E during incubation at a fixed ATP concentration (40 µM) is depicted in Fig. 4A by comparing inactivation of the enzymes in Groups 1, 2, and 3. Group 1 (wild-type E, MS2, MS3, and MS2,3) represents phosphorylation mostly of site 1 in the presence or absence of sites 2 and 3; Group 2 (MS1 and MS1,3) represents phosphorylation of site 2 in the absence or presence of site 3, and Group 3 (MS1,2) shows phosphorylation of site 3 only. The inactivation of the enzymes of Group 1 was complete after 10 min incubation, Group 2 was inactivated in 30 min, while Group 3 having only site 3 required more than 100 min for complete inactivation. Results presented in Fig. 4showed that phosphorylation with concomitant inactivation of the enzymes was found for each of the three sites, but the rates of these processes were different for each site.

The initial rates of phosphorylation and inactivation of wild-type E and the mutant Es are compared in . The rates were similar for all species having site 1 (Group 1). Group 2 (without site 1 but with site 2 available) showed slower rates of phosphorylation and inactivation than Group 1. The lowest rates of phosphorylation and inactivation were found for Group 3 with only site 3 available.

Phosphorylation of E Mutants by the E Kinase in the Absence of E

Fig. 5shows the incorporation of P and inactivation of wild-type E and E mutants during incubation with E-kinase and ATP. To achieve phosphorylation of E by the E-kinase in the absence of E it was necessary to increase the concentrations of both the E-kinase and ATP. Complete inactivation of wild-type E occurred after 15 min incubation in the presence of 200 µM ATP. Mutants MS3, MS2, MS2,3 were phosphorylated with concomitant inactivation similar to wild-type E indicating that site 1 can be phosphorylated in the absence of E. However, mutants having only site 2 (MS1,3), site 3 (MS1,2), or sites 2 and 3 (MS1) exhibited low levels of phosphorylation (10-15% of that for wild-type E and mutants with site 1), and low levels of inactivation (approximately 10% of that for wild-type E and mutants with site 1). Autoradiography (Fig. 5C) showed a low level of phosphate incorporation into sites 2 and 3, when only the E-kinase was present, and this was much less than phosphorylation of the Es by the E-X-kinase subcomplex (Fig. 4C).


Figure 5: A, inactivation of Es by phosphorylation by the E-kinase in the presence of 200 µM ATP. Activity was measured in the reconstitution assay. 100% activity for each curve corresponded to the activity of each E at zero time. B, P incorporation from [-P]ATP (200 µM) into Es by the E-kinase. Seven µg of the E and 2 µg of the E-kinase were used in each reaction. Incorporation of P was measured by scintillation counting of paper discs. Phosphorylation in control tubes containing only the E-kinase (without E) was subtracted. C, autoradiography of E after incorporation of -P followed by separation of proteins in SDS-PAGE. Upper band represents autophosphorylation of the E-kinase, and the lower band represents P incorporation into E subunit.



shows the initial rates of incorporation of P and inactivation during phosphorylation of Es by the E-kinase. The difference between the rates of inactivation and phosphorylation for sites 2 and 3 compared to that of site 1 was much larger during phosphorylation by the E-kinase alone than during phosphorylation by the E-kinase in the presence of E. The corresponding k of inactivation (and rates of phosphorylation) were: in the absence of E for site 1, 0.347 min (0.125 mol of P/mol of E); for site 2, 0.002 min (0.005 mol of P/mol of E); for site 3, 0.003 min (0.007 mol of P/mol of E); and in the presence of E for site 1, 0.732 min (0.241 mol of P/mol of E); for site 2, 0.096 min (0.053 mol of P/mol of E); and for site 3, 0.022 min (0.015 mol of P/mol of E).

Dephosphorylation of E Mutants by the Phospho-E-phosphatase

Fig. 6shows the reactivation of the phosphorylated wild-type and mutant Es during dephosphorylation by the phospho-E-phosphatase. All Es with sites 1, 2, and 3 present either individually or in combination with the other sites were dephosphorylated by the phospho-E-phosphatase (Fig. 6B) with similar rates of P release as reflected in . The calculated rates of dephosphorylation for sites 1, 2, and 3 were: 0.111 min, 0.104 min, and 0.102 min, respectively. Dephosphorylation resulted in complete reactivation of all the mutant enzymes (Fig. 6A). The rates of reactivation were similar for the E proteins with only site 1 (MS2,3), site 2 (MS1,3), or site 3 (MS1,2) (Fig. 6A, inset; also see ). The reactivation was slower for the mutants with two phosphorylation sites (MS1, MS2, MS3) than the mutants with only one site (MS2,3, MS1,3, MS1,2), and the reactivation was slowest for E with three potential phosphorylation sites than for the mutants with two sites (). The reactivation curves for the mutants with two sites and especially for wild-type E were sigmoidal, and longer incubation times were necessary to achieve complete reactivation. The different behavior of the mutants with different number of phosphorylation sites can be explained by the fact that activity depends upon dephosphorylation of all of the available sites.


Figure 6: A, reactivation of Es during dephosphorylation by the phospho-E-phosphatase. Activity was measured in the reconstitution assay. Inset shows the semilog plots of reactivation of MS2,3, MS1,3, and MS1,2 by the phospho-E-phosphatase. Ao, inactivation (%) at zero time; At, inactivation (%) at time intervals indicated. This is a representative experiment of the results presented as the means ± S.E. (n = 4) shown in Table II. B, P release from phosphorylated Es by the phospho-E- phosphatase. E proteins were phosphorylated by the E-X-kinase in the presence of 50 µM [-P]ATP for 45 min. Ten µg of the phosho-E and 1 µg of the phospho-E-phosphatase were used in each dephosphorylation reaction. P release was determined by taking aliquots at different time intervals, protein precipitation with trichloroacetic acid, and measuring the radioactivity by counting the soluble fraction in a scintillation counter. The level of radioactivity released with acid treatment at zero time (before the phospho-E-phosphatase addition) was subtracted from each point. C, the semilog plots of P release from phosphorylated MS2,3, MS1,3, and MS1,2 by the phospho-E-phosphatase. Bo, P bound with the protein (nmol/mg E) at zero time; Bt, P bound with the protein (nmol/mg E) at time intervals indicated. This is a representative experiment of the results presented as the means ± S.E. (n = 5) shown in Table II.




DISCUSSION

Phosphorylation of key regulatory enzymes is one of the major mechanisms for regulation of metabolic processes. PDC catalyzes the decarboxylation of pyruvate and provides acetyl-CoA for the generation of ATP and also for the synthesis of fatty acids, cholesterol, and some amino acids. Because of the critical role played by this multienzyme complex in carbohydrate metabolism, its activity is modulated by the metabolic state of the cell. One of the major mechanisms of regulating the activity of PDC is by reversible phosphorylation-dephosphorylation of the E subunit catalyzed by the E-kinase and the phospho-E-phosphatase, respectively. The activities of these two enzymes are regulated by the concentration ratios of ATP/ADP, NAD/NADH, acetyl-CoA/CoA, and by pyruvate, TPP, Mg, and Ca(2, 22) .

Three serine residues were identified by protease digestion of phosphorylated E from bovine and porcine PDC (11, 23). These three serine residues are referred to as site 1, site 2, and site 3 that correspond to Ser-264, Ser-271, and Ser-203 in the human E sequence, respectively(24) . It was shown that phosphorylation of site 1 caused the major inactivation of the enzyme(11, 25) . However, the role of sites 2 and 3 in enzyme inactivation is not well understood(26, 27, 28) . Also, it is not clear whether sites 2 and 3 can undergo phosphorylation in the absence of site 1. It was suggested that sites 2 and 3 are important for dephosphorylation and reactivation of E by the phospho-E-phosphatase(28, 29) . As all three sites were shown to undergo phosphorylation at different rates, it was difficult to determine the possible role of sites 2 and 3 in the presence of the highly reactive site 1(23, 26) .

In the present study we took advantage of site-directed mutagenesis and made E mutants having only site 1 (MS2,3), site 2 (MS1,3), site 3 (MS1,2) or all possible combinations of two sites. Using these mutant enzymes it was possible to study phosphorylation of each site separately or in combination with the other sites and determine the extent of inactivation. All the mutants having one or two phosphorylation sites were phosphorylated by the E-kinase bound with E. This shows a random rather than sequential mechanism of phosphorylation. Phosphorylation of each site alone resulted in enzyme inactivation. In an earlier study only 0.7-6.4% of inactivation was suggested for site 2 phosphorylation, and phosphorylation at site 3 was considered to be non-inactivating(26) . Although the phosphorylation of a single site resulted in complete inactivation, the rates of phosphorylation and inactivation were site-specific. High rates were observed for the E mutants having site 1 alone or in combination with site 2 or 3 (Group 1: MS2,3, MS2, MS3); low rates were observed for the E mutants having site 2 alone or with site 3 (Group 2: MS1, MS1,3), and very low rates were detected for the mutant with site 3 alone (Group 3: MS1,2) (). Different rates of phosphorylation were proposed earlier based on determining site occupancy by tryptic digestion and high-voltage paper electrophoresis(30) .

The rates of dephosphorylation of the three sites and possible involvement of sites 2 and 3 in dephoshorylation of site 1 have remained controversial(27, 28, 29) . In previous studies, the dephosphorylation rates of individual sites were determined by measuring the radioactivity at each site in tryptic phosphopeptides generated from the partially and fully phoshorylated E proteins(27, 28) . Using this approach the relative rate of dephoshorylation was found to be higher for site 2 than site 3, and also higher for site 3 than site 1(27, 28) . Also, the rate of dephosphorylation of the fully phosphorylated E (three site occupancy) was higher than that of a partially phosphorylated enzyme(27, 28, 29) . Using selective thiophosphorylation of sites 2 and 3 (which remained resistant to hydrolysis by the phospho-E-phosphatase), it was shown that the modification of sites 2 and 3 did not affect the rate of dephosphorylation of site 1 of bovine E, suggesting a random dephosphorylation of the three sites(27) . In contrast, reactivation of phosphorylated porcine E was found to be faster for the partially phosphorylated enzyme than for the fully phosphorylated E(28, 29) . Based on these findings it was proposed that the mechanism of dephoshorylation is not purely random and that dephosphorylation of site 1 in fully phosphorylated E is not independent of that of site 2(28) .

Availability of the site-specific human E mutants has allowed us to examine possible interactions among the three sites in dephoshorylation. We observed that the mutant phospho-Es having only one site available for phoshorylation (e.g. sites 1, 2, or 3) displayed similar rates of dephosphorylation as well as reactivation (Fig. 6, ). Furthermore, the rates of dephosphorylation of the E mutant proteins with only two sites available for phosphorylation were similar to that of E proteins with either any one site or all three sites available for phosphorylation (Fig. 6, ), supporting the earlier finding of a random mechanism for dephosphorylation of the three sites. Although we did not measure the rate of dephosphorylation of each site in the mutant Es (MS1, MS2, and MS3) having any two sites present, the observed similarity in the dephosphorylation rates of the three E mutants with any two phosphorylation sites intact (MS1, MS2, MS3) indicates that the presence of any one site did not influence dephosphorylation of the other site present (Fig. 6). The reactivation of the different mutant Es during dephosphorylation was different, however. The reactivation of the E mutants with only one site phosphorylated was faster than that of the E mutants with any two sites which in turn was faster than that for the E with three sites. These differences in the reactivation rates can be explained by the presence of two instead of one site (or three instead of two sites) which are required to be dephosphorylated to activate the enzyme. As shown above, phoshorylation of any one site results in inactivation of the enzyme (Fig. 4). Our findings clearly indicate that sites 1, 2, and 3 are dephosphorylated independently of each other.

There was no indication of interactions among sites 1, 2, and 3 in the E subunit. Individual sites seemed to behave the same either in the presence or absence of other sites. However, phosphorylation of one Ser residue (Ser-264, Ser-271, or Ser-203) per E molecule was enough for complete inactivation of the enzyme. We found that the half-of-the-site reactivity is true not only during phosphorylation of site 1 as was shown earlier(11, 25) , but is also the case during phosphorylation of sites 2 or 3. Maximal level of incorporation in different experiments with two preparations of the enzymes was not more than 1 mol of P/mol of E for mutants with one site available, not more than two for mutants with 2 sites present while maximum number of phosphoryl groups incorporated in E was not more than three which corresponds to the previously reported data on highly purified porcine PDC(25) . This was suggested previously that three phosphoryl groups can be incorporated in one -subunit(25) . It is possible if one takes into consideration that tetrameric E is a highly cooperative enzyme, showing interaction between its two active sites during its interaction with the substrate and cofactor, and also during catalysis(31) . Two active sites of this enzyme were proposed to display an alternating site catalytic mechanism where an event in one active site depends upon the reaction catalyzed in the other active site(31) . It has half-the-site reactivity when interacting with HETPP (32). The intersubunit interactions are revealed also in the different reactivities of essential amino acid residues, belonging to the neighboring active site(31) . The kinetic model, including a conformational transition from one active site after its modification to the other has been proposed by Khailova et al.(31) . The existence of an interaction between the two active sites can explain the observed regulation by phosphorylation of one of the two -subunits by the E-kinase.

The phosphorylation of the three serine residues of E subunit was found to be different when catalyzed by the E-kinase not bound to E(11, 30) . We confirmed this observation by using the phosphorylation site E mutants. We observed that site 1 undergoes phosphorylation by the E-kinase only leading to its inactivation, but this requires higher ATP and kinase concentrations compared to phosphorylation of the site 1 in E by the E-kinase in the presence of the E. For sites 2 and 3 phosphorylation and inactivation were much lower ( Fig. 6and ). This phenomenon has been attributed to a conformational change induced in E or E-kinase after association with the E(30) .

Recent studies of the phosphorylation site 1 (Ser-293) and site 2 (Ser-303) of the branched-chain -ketoacid dehydrogenase complex (33) by site-directed mutagenesis showed that phosphorylaton of only site 1 or mutation of serine 293 to glutamate caused enzyme inactivation. However, phosphorylation of site 2 or replacement of serine 303 to glutamate did not effect enzyme activity, indicating non-involvement of the site 2 with respect to regulation of the activity by phosphorylation. This contrasts with our findings on PDC E, as phosphorylation of sites 2 and 3 caused complete inactivation of E. This specificity may reflect the differences between the amino acid sequences surrounding sites 1 and 2 of the two multienzyme systems.

What could be the function of three phosphorylation sites when phosphorylation of each of them is sufficient for complete inactivation of E (and hence the whole multienzyme complex)? It is plausible that since the metabolism of pyruvate via the PDC is a critical step in maintenance of glucose homeostasis that the presence of multiple sites (e.g. sites 2 and 3) is developed as a safety mechanism for regulation of the complex activity if an unexpected mutation occurs at site 1.

A serine to alanine mutation used in the present study does not represent a significant change in the size of the amino acid side group, and only a change in the polarity and hydrophobicity of this residue. Nevertheless such a change at Ser-264 (site 1) decreased the specific activity of the enzyme to 40-50% of the wild-type enzyme and caused increases in K values for TPP and pyruvate (). This suggests that either site 1 is closer to the active site than the other sites; or that it lies on the pathway of the main catalytic conformational change. The activities of E measured by the DCPIP and CO assays were also decreased for E with mutation at site 1 to the same extent as compared to activity measurement after reconstitution of E with the E-X and E. This finding excludes the possibility of the S264A mutation affecting the interaction of E with E.

The mechanism of E inactivation by phosphorylation is not well understood. It was found that phosphorylation of pig heart E inhibited all the partial reactions (forward and backward) leading to the formation of HETPP(12) . Butler et al.(34) showed that phosphorylation affected TPP binding to E, but this effect was not significant since the transition state analog of TPP, thiamine thiazolone pyrophosphate, could interact with phosphorylated E. It was suggested that phosphorylation produced a conformational change in E that displaced a catalytic group (or groups) at the active site(34) . Spectral studies of pigeon breast muscle phospho-E showed that it bound TPP in the active conformation with the formation of a charge transfer complex, to interact with HETPP by the alternating site mechanism, but could not bind the substrate pyruvate(13) . It was suggested that a negative phosphoryl residue may compete for the active site arginine and thereby block the substrate binding. Phosphorylation of a serine residue has been shown to regulate the activity of other enzymes in different ways: (i) enhancing the activity of glycogen phosphorylase by means of a long-range conformational change upon phosphorylation(35) ; (ii) abolishing isocitrate dehydrogenase activity as a result of electrostatic repulsion and steric hindrance between the phosphoryl moiety and the carboxylic group of the substrate(36) ; and (iii) attenuating the activity of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase by impairing the ability of the catalytic histidine to protonate CoA(37) . Which one of these possibilities is attributed to phosphorylation and hence inactivation of E remains to be determined.

  
Table: K values for pyruvate and TPP for E and phosphorylation site mutants


  
Table: Rates of phosphorylation and inactivation of E and its mutants by the E-X-kinase subcomplex or by the E-kinase alone and of dephosphorylation and reactivation by the phospho-E-phosphatase



FOOTNOTES

*
This work was supported by United States Public Health Service Grant DK20478. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, State University of New York at Buffalo, 140 Farber Hall, 3435 Main St., Buffalo, NY 14214.

The abbreviations used are: E, pyruvate dehydrogenase; E, dihydrolipoamide acetyltransferase; E, dihydrolipoamide dehydrogenase; PDC, pyruvate dehydrogenase complex; E-X-kinase, dihydrolipoamide acetyltransferase-protein X-E kinase; TPP, thiamin pyrophosphate; HETPP, 2-hydroxyethylidene-thiamin pyrophosphate; Ni-NTA-agarose, Ni-nitrilotriacetic-agarose; DCPIP, 2,6-dichlorophenolindophenol; PAGE, polyacrylamide gel electrophoresis.

M. S. Ali, B. C. Shenoy, D. Eswaran, L. A. Andersson, T. E. Roche, and M. S. Patel, unpublished data.

D. Eswaran, M. S. Ali, B. C. Shenoy, L. G. Korotchkina, T. E. Roche, and M. S. Patel, unpublished data.

T.-C. Liu, L. G. Korotchkina, S. Hyatt, N. N. Vettakkorumakankav, and M. S. Patel, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Thomas E. Roche for preparation of highly purified bovine kidney E, E-protein X-kinase subcomplex, and phospho-E-phosphatase. We are indebted to Dr. Robert A. Harris for a preparation of recombinant E-kinase. We are grateful to Drs. Edward Niles, Murray Ettinger, and Cecile Pickart of this department for critical reading of the manuscript.


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