©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Tryptophan Residue in the Thiamin Pyrophosphate Binding Site of Mammalian Pyruvate Dehydrogenase (*)

(Received for publication, November 18, 1994; and in revised form, December 12, 1994)

M. Showkat Ali (1) (2) Bhami C. Shenoy (1) Devayani Eswaran (1) Laura A. Andersson (3) Thomas E. Roche (3) Mulchand S. Patel (1) (2)(§)

From the  (1)Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, the (2)Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214, and the (3)Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The pyruvate dehydrogenase (E1) component of the mammalian pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate with the formation of an acetyl residue and reducing equivalents, which are transferred sequentially to the dihydrolipoyl acetyltransferase and dihydrolipoamide dehydrogenase components. To examine the role of tryptophanyl residue(s) in the active site of E1, the enzyme was modified with the tryptophan-specific reagent N-bromosuccinimide. Modification of 2 tryptophan residues/mol of bovine E1 (out of 12 in a tetramer alpha(2)beta(2)) resulted in complete inactivation of the enzyme. The inactivation was prevented by preincubation with thiamin pyrophosphate (TPP), indicating that the modified tryptophan residue(s) is a part of the active site of this enzyme. Fluorescence studies showed that thiamin pyrophosphate interacts with tryptophan residue(s) of E1. The magnetic circular dichroism (MCD) spectral intensity at 292 nm was decreased by 15% for E1 + TPP relative to the intensity for E1 alone. Because this MCD band is uniquely sensitive to and quantitative for tryptophan, the simplest interpretation is that 1 out of 6 tryptophan residues present in E1 (alphabeta dimer) interacts with TPP. The natural circular dichroism (CD) spectrum of E1 is dramatically altered upon binding TPP, with concomitant induction of optical activity at 263 nm for the nonchiral TPP macrocyle. From CD studies, it is also inferred that loss of activity following N-bromosuccinimide treatment occurred without significant changes in the overall secondary structure of the protein. A single peptide was isolated by differential peptide mapping in the presence and absence of thiamin pyrophosphate following modification with N-bromosuccinimide. This peptide generated from human E1 was found to correspond to amino acid residues 116-143 in the deduced sequence of human E1beta, suggesting that the tryptophan residue 135 in the beta subunit of human E1 functions in the active site of E1. The amino acid sequences surrounding this tryptophan residue are conserved in E1beta from several species, suggesting that this region may constitute a structurally and/or functionally essential part of the enzyme.


INTRODUCTION

The multienzyme pyruvate dehydrogenase complex (PDC) (^1)plays a pivotal role in energy metabolism by regulating the oxidation of pyruvate. The mammalian complex consists of several catalytic as well as regulatory components namely, pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase, dihydrolipoyl dehydrogenase, protein X, E1alpha-kinase, and phospho- E1alpha-phosphatase(1, 2) . The E1 tetramer (alpha(2)beta(2)) has two active centers with equal catalytic efficiency but different affinity for thiamin pyrophosphate (TPP)(3) . Considering the importance of this component in the overall catalytic reaction in the complex, little is known about the structure of the active site. Studies involving chemical modification of E1 have implicated the amino acid residues lysine(4) , histidine, arginine, tryptophan, and cysteine (5) as being critical for enzyme function. The involvement of tryptophan in TPP binding was suggested by chemical modification of pigeon breast E1 using N-bromosuccinimide (NBS)(6) .

The primary structures of both alpha and beta subunits of E1 from several species have been deduced from their cDNA sequences(7, 8, 9, 10, 11) . The possible functional significance of several motifs in these deduced sequences have also been proposed based on sequence homology(12, 13) . Hawkins et al. (12) have identified a sequence motif of approximately 30 residues in length in several TPP-dependent enzymes; this motif begins with the sequence GDG and ends with NN. Aspargine has also been suggested as a possible residue for TPP binding(12) . Dahl et al. (14) have speculated that tryptophan residue 214 of the alpha subunit of human E1 may be a possible site of TPP binding. In a number of TPP-dependent enzymes like Escherichia coli pyruvate oxidase(15) , Zymomonas mobilis pyruvate decarboxylase(16) , and Saccharomyces cerevisiae transketolase(17) , tryptophan has been implicated in TPP binding from spectral analysis and chemical modification studies. Thus, clarification of the involvement of tryptophan or aspargine residue(s) in TPP binding in these enzymes is of fundamental importance.

To investigate the possible role of tryptophan residue(s) in the structure-function relationship and to explore its specific localization in the E1 component of mammalian PDC, we have treated both highly purified bovine E1 and recombinant human E1 with NBS, a tryptophan-specific chemical reagent. We have identified by differential peptide mapping and reverse phase high performance liquid chromatography (HPLC) followed by sequence analysis that the tryptophan residue of E1, which is involved in TPP binding, corresponds to the tryptophan residue at position 135 in the human E1beta subunit.


EXPERIMENTAL PROCEDURES

Materials

TPP, sequencing grade trypsin, and pyruvate were purchased from Sigma; NBS was purchased from Aldrich; HPLC grade acetonitrile was from Fisher, and trifluoroacetic acid was from Pierce.

Assay of E1 Activity

Bovine kidney PDC was purified as previously described(18) , and its components were resolved according to Linn et al.(19) . Recombinant human E1 overexpressed in E. coli M15 cells was purified by the method of Korotchkina et al.(20) . PDC activity was assayed by reconstituting E1 with dihydrolipoyl acetyltransferase-X and dihydrolipoyl dehydrogenase according to the method of Roche and Reed(21) . Protein was determined spectrophotometrically using BIO-RAD protein reagent, using bovine serum albumin as the standard.

Modification of E1 with NBS

Bovine E1 (0.64 nmol) was incubated with varying concentrations of freshly prepared NBS in 0.1 ml of 20 mM potassium phosphate buffer (pH 6.5) at 25 °C. The progress of the reaction was monitored at 280 nm (21) in a Shimadzu spectrophotometer (model UV160U), while the corresponding decreases in enzymatic activity were measured by analyzing aliquots from reaction mixtures directly in the standard reconstituted PDC activity assay(21) . The number of tryptophan residues oxidized by NBS was determined by the method of Spande and Witkop(22) . The possibility of modification of tyrosine residues was also assessed from spectral analysis(23) . In the protection experiments, the enzyme was preincubated with TPP alone, pyruvate alone, or TPP plus pyruvate, for 10 min followed by incubation with varying concentrations of NBS.

Fluorescence Measurement

Fluorescence measurements were carried out with a Perkin-Elmer (model LS-5B) spectrofluorometer with an excitation wavelength of 295 nm (bandwidth, 10 nm). The emission spectra were obtained over the range of 300-400 nm (bandwidth, 5 nm). Reactions were performed at 25 °C and initiated by addition of incremental amounts of TPP to bovine E1 (0.1 mg/ml) in 20 mM potassium phosphate buffer, pH 6.5.

Circular Dichroism Spectral Analyses

MCD and circular dichroism (CD) spectra were obtained for bovine E1 in the presence of a 2-fold excess of TPP and also for a 200 µM sample of TPP itself. The data were obtained on Jasco J-720 spectrometer, equipped with a 1.5 Tesla (15,000 Gauss) electromagnet, using 1-mm quartz suprasil cuvettes. The instrument was calibrated daily prior to use with ammonium-d camphor-10-sulfonate (Jasco, Tokyo). Measurements were carried out at 18 °C under a magnetic flux density of 1.39 Tesla, with the magnetic field direction parallel to the direction of light propagation.

All samples were in pH 7.2 potassium phosphate buffer containing 1 mM Mg-EDTA. Experimental conditions for data collection were: wavelength range, 250-350 nm; 9-16 scan accumulation; spectral step resolution, 0.1 nm; scan rate, 20 nm/min; slit width, 1 nm; response, 0.25 s. Data manipulation and calculations utilized the Jasco J-720 software. CD data are presented as Delta (M cm). Far ultraviolet (protein region CD spectra of both native and NBS-modified E1 preparations were recorded between 190 and 260 nm at 25 °C using a J-600 Spectropolarimeter (Jasco, Tokyo).

Protease Digestion and Reverse Phase HPLC Separation of Peptides

Human E1 (6.4 µM) modified with 0.25 mM NBS both in the presence and absence of TPP (1 mM) and the reaction was quenched by the addition of 10-fold excess of tryptophan. The samples were dialyzed extensively overnight against 50 mM potassium phosphate buffer (pH 7.5) and then digested with trypsin (1:25, w/w, protease:protein) for 20-24 h in the presence of 6 M urea. In the case of NBS-modified bovine E1, the protein was digested with V8 protease for 48 h in the presence of 2 M guanidinium chloride. The reactions were stopped with the addition of 0.1% trifluoroacetic acid. Separation of peptides generated after trypsin digestion of both native and NBS-oxidized E1 samples was achieved using a Shimadzu HPLC system (model LC 600) equipped with a Synchropack C-4 reverse phase column (4.6 times 25 mm). The column was equilibrated with 0.1% trifluoroacetic acid in triply distilled water, and peptides were eluted with a 0-60% acetonitrile gradient containing 0.1% trifluoroacetic acid over a period of 120 min at a flow rate of 0.9 ml/min. Absorbance was monitored simultaneously at 220 and 280 nm. The peptide peak of interest was dissolved in 0.5 ml of potassium phosphate buffer, pH 7.5, and reinjected onto a C-8 reverse phase column. Separation was performed using a linear gradient of 10-40% acetonitrile in 90 min at a flow rate of 0.9 ml/min. The purified peptide was sequenced using an Applied Biosystems model 470A/120A automatic gas phase sequencer(24) .


RESULTS AND DISCUSSION

Intrinsic Tryptophanyl Fluorescence of E1

Bovine E1 exhibited a characteristic fluorescence emission with maximal intensity at 335 nm when excited at 295 nm. At this excitation wavelength, both tyrosine and phenylalanine contribute very little to the observed fluorescence, and the inner filter effect due to TPP absorption is also minimized. Experiments were conducted to study whether TPP, pyruvate, or Mg interactions with the enzyme would induce changes in the accessibility of tryptophan residue(s) as reflected by changes in fluorescence quenching patterns. Among these ligands, only TPP substantially quenched the fluorescence intensity of E1. Fig. 1A shows that sequential addition of TPP to bovine E1 produced a progressive quenching of the tryptophanyl fluorescence of E1. Fig. 1B shows the dependence of the fluorescence intensity at the emission maximum on TPP concentration. These data indicate that the fluorescent tryptophan residue(s) is located at or near the TPP binding site.


Figure 1: A, effect of TPP on the tryptophanyl fluorescence emission spectrum of bovine E1. Varying concentrations of TPP were added to E1 (0.1 mg/ml), and emission spectra were collected from 300-400 nm after excitation at 295 nm. B, plot of relative fluorescence intensity of E1 at the emission maximum (335 nm) as a function of concentration of TPP.



Circular Dichroism Spectral Analyses of Bovine E1

Barth et al. (25) first reported the use of MCD spectroscopy for quantitative determination of tryptophan, noting that ``tryptophan is the only naturally occurring amino acid that gives a positive MCD band.'' The 292-nm MCD band intensity for tryptophan was shown to be independent of the amino acid location within the peptide chain and was not influenced by peptide conformation, even upon protein denaturation. Furthermore, the MCD intensity of tryptophan was not affected by pH in the range between 1 and 12(25) . As noted by Holmquist and Vallee(26) , MCD spectroscopy can be used with high sensitivity and accuracy to identify and quantitate tryptophan.

Fig. 2presents the MCD spectra of bovine E1 + TPP (solidline), E1 (dashedline), and the (E1 + TPP) - E1 difference spectrum (dotted-dashed line). The MCD maximum is, as expected, at 292 nm for both native and the (E1 + TPP) samples. A 15-17% decrease in intensity, concomitant with a red shift of 0.3 nm, occurred upon binding of TPP to bovine E1 (compare the solid and dashedspectra). This is perhaps most clearly shown by the dotted-dashed line, the difference spectrum of (E1 + TPP) - E1. These data strongly suggest that 1 out of 6 tryptophan residues of E1 interacts with TPP leading to a major loss in its MCD signal. We cannot eliminate partial reduction of the signal of more than one Trp, but the reduction in signal must result from an interaction changing the electronic character of Trp, and this is most easily explained with a change in single Trp.


Figure 2: MCD spectra of bovine E1. Dashedline, E1 (45.5 µM) in potassium phosphate buffer (pH 7.2) and 1 mM Mg-EDTA; solidline, E1 (43.7 µM) plus TPP (87.4 µM) in same buffer; dotted-dashed line, difference spectrum ((E1 + TPP) - E1). Data were obtained and calculated using the J-720 software.



One possibility for the loss in signal is that binding of TPP to E1 involves a ``stacking'' interaction with the tryptophan residue(5) . It is interesting, if this model is accurate, that stacking should cause a loss of MCD intensity (to our knowledge, such a perturbation of tryptophan MCD by stacking has not been previously addressed). The MCD bands of tryptophan were identified by comparision with their absorption bands (assigned by Strickland et al.(27) ). The 292-nm MCD band of indole chromophores, i.e. tryptophan, was assigned by Holmquist and Vallee (26) to a -*, L(b) (0-0) transition. Albinsson et al. (28) showed that the UV transition moment was at + 45 ± 5^o relative to the pseudo symmetry long axis. Thus, our data suggest that the nature of the tryptophan-thiamin interaction results in loss of this transition. This is of particular interest given the normal insensitivity of this feature to perturbants such as solvent, pH, or protein conformation.

Fig. 3presents the natural CD spectra of bovine E1 + TPP (solidline), E1 (dashedline), and TPP alone (parallelingbaseline). As evident from the spectrum, TPP alone has no optical activity. However, upon binding to bovine E1, a dramatic spectral change occurred (compare solid and dashedspectra). The entire spectrum becomes shifted upwards, and there is a new, intense feature at 263 nm, coincident with the electronic absorption maximum of TPP. Thus, these data represent a classic example of ``induced optical activity,'' with the optically inactive TPP coupling to the optically active protein chromophore, resulting in the observed spectral pattern. There is also a dramatic shift at higher wavelengths, as previously reported by Khailova and co-workers(3, 5) .


Figure 3: CD spectra of bovine E1. Dashedline, E1 (45.5 µM) in potassium phosphate buffer (pH 7.2) and 1 mM Mg-EDTA (pH 7.2); solidline, E1 (43.7 µM) + TPP (87.4 µM) in the same buffer; and lineparallelingbase line, TPP (200 µM). CD spectra are presented as Delta (M cm), where = 3300Delta.



NBS Oxidation of E1

Incubation of bovine E1 with increasing concentrations of NBS resulted in a progressive decrease in the enzyme activity (Fig. 4A). About 95% of the activity was lost from the treatment of E1 with 0.25 mM NBS for 1 min. Addition of TPP provided protection against NBS inactivation; about 60% protection was afforded by 0.4 mM TPP. However, a further increase in concentration of TPP to 1 mM did not have any significant additional effect. A similar effect of TPP against the inactivation of E. coli pyruvate oxidase has also been reported(15) . Addition of pyruvate to bovine E1 did not protect against NBS inactivation. Our findings of TPP protection against NBS inactivation of bovine E1 also agree with the results of Khailova et al.(5) on pigeon breast E1. Thus, location of tryptophan residue(s) at or close to the TPP binding site may be a general feature of the active site of PDC E1s from higher organisms. Interestingly, E1 was not protected from inactivation when both TPP and pyruvate were added simultaneously. The formation of the charge transfer complex in the presence of TPP alone affords the protection against NBS modification of E1. The addition of pyruvate to holo-E1 has been shown to result in the disappearance of the charge transfer complex(5) , which may explain the elimination of protection.


Figure 4: A, inactivation of E1 with NBS. E1 (6.4 µM) in 20 mM potassium phosphate buffer (pH 6.5) was preincubated in presence and absence of TPP and/or pyruvate and then treated with varying concentrations of NBS for 1 min and expressed as NBS/E1 molar ratio. Treatments were E1 (box), E1 preincubated with 0.4 mM TPP (circle) and with 1 mM TPP (), E1 preincubated with 0.05 mM TPP plus 0.5 mM pyruvate (up triangle), and E1 preincubated with 0.5 mM pyruvate (bullet). B, correlation between the oxidation of tryptophan residue(s) () in E1 and absorbance (up triangle) with the NBS/E1 molar ratio. The number of tryptophan residues oxidized was calculated from the decrease in absorbance at 280 nm.



The oxidation of bovine E1 with NBS produced an instantaneous decrease in absorbance at 280 nm with the transformation of the indole to oxindole chromophore (Fig. 4B). The maximal change in absorbance during the NBS titration can be used to determine the number of tryptophan residues oxidized(22) . Fig. 4B shows the concentration dependence for NBS modification of tryptophan residues in E1 and supports a stoichiometric reaction. At relatively low NBS/E1 molar ratio (40:1), 2 tryptophan residues/mol of E1 (alpha(2)beta(2)) were oxidized. Based on the amino acid sequences of human E1alpha and E1beta subunits, there are a total of 12 tryptophan residues in the tetrameric E1 (2 in alpha and 4 in beta). Only 2 of the 12 tryptophan residues were modified by NBS under our experimental condition. Thus, a single tryptophan residue per subunit of bovine E1 appeared to be at or near the catalytic site. The number of essential tryptophan residues oxidized also correlates with the number of active centers of the enzyme(3) . Table 1provides results showing the extent of tryptophan modification in the absence and presence of different ligands. TPP (1 mM) alone caused almost complete protection of two tryptophan residues, but other combinations were ineffective in protecting the tryptophan residues from NBS modification (Table 1). The CD spectrum in the far ultraviolet region of the modified enzyme was essentially identical with that of the untreated enzyme (results not shown), indicating little or no significant change in secondary structure of the protein upon modification.



Identification of Tryptophan Residue(s) Involved in TPP Binding

Our data using bovine E1 indicate that a single tryptophan residue per subunit (either in alpha and beta) of E1 is protected by TPP. To isolate the peptide containing the active site tryptophan residue, recombinant human E1 was treated with NBS (0.25 mM) under similar conditions as were used for bovine E1. The extent of modification of tryptophan residue was quantitated from the decrease in absorbance at 280 nm. Complete loss of activity again correlated with the modification of a single active site tryptophan residue. TPP (0.4-1 mM) afforded 60-70% of protection against NBS modification of human E1 (results not shown). To locate this Trp residue, we employed differential peptide mapping following NBS inactivation of the human E1 in the presence and absence of TPP. After NBS modification, the enzyme was digested with trypsin, and the peptides were separated by reverse phase HPLC. The eluant from the HPLC column was monitored simultaneously at 220 and 280 nm. A typical chromatogram monitored at 280 nm is shown in Fig. 5. Detailed inspection of the chromatogram indicates that the peptide marked by plus sign eluting at 30% acetonitrile (at 60 min) was substantially protected by TPP. Other peptide peaks did not show any pronounced changes in the presence or absence of TPP. Since 2 mol of tryptophan were modified per mole of tetrameric E1 (Fig. 4B), the recovery of one tryptophanyl peptide as obtained is in accordance with the data above. The tryptophanyl peptide peak was collected and further purified by rechromatography on C-8 reverse phase column. This HPLC run provided only one major peak at 280 and 220 nm (results not shown). The peptide was concentrated and then sequenced.


Figure 5: Reverse phase HPLC elution profile of tryptic digest of human E1. Absorbance was monitored at 280 nm for the detection of tryptophanyl peptides. The profiles correspond to E1 modified with NBS (0.25 mM) in the presence of TPP (1 mM) (A) and E1 modified with NBS (0.25 mM) in the absence of TPP (B). The plussign indicates the peak of interest as described under ``Results and Discussion.'' Experimental details were as described under ``Experimental Procedures.''



The peptide was expected to be very long, as judged from an HPLC elution profile and from the predicted trypsin cleavage sites. The peptide was sequenced for the first 15 amino acid residues. The sequence GPNGASAGVAAQHSQ matched completely with the sequence from 116 to 130 amino acid residues in human E1beta, indicating tryptic cleavage occurred after Arg-115 in this protein. The next predicted trypsin cleavage site would be at Lys-143, giving rise to a peptide of 28 amino acid residues with only 1 tryptophan at 135. By a similar differential procedure, a peptide with the sequence AWYG was isolated from bovine E1 following V8 protease digestion. The sequence agrees with that around the same Trp in the human E1beta subunit (cf. Fig. 6, discussed below). Although we are unable to explain the production of this peptide by V8 protease, this further supports the conclusion that it is this specific Trp of the E1beta subunit, which is protected from modification by TPP. These results strongly support the contention that Trp-135 of E1beta is involved in TPP binding.


Figure 6: Comparison of the amino acid sequences surrounding the specific tryptophan residue in the beta subunit of PDC-E1 from recombinant human E1 (H*, present study), human (H)(8) , rat (R)(9) , pig (P)(10) , nematode (N)(11) , and Bacillus stearothermophillus (B)(29) .



Amino acid sequences of the E1beta subunit of PDC have been deduced from cDNAs isolated from human(8) , rat(9) , pig(10) , nematode(11) , and Bacillus stearothermophillus(29) . Comparison of the aligned sequences of these E1beta subunits indicates that Trp-135 in human E1beta is conserved among these proteins; amino acid residues surrounding the modified tryptophan are highly conserved (Fig. 6). Thus, this region may constitute a part of the enzyme active site.

Because it has not been possible to isolate functionally active E1alpha and E1beta separately, it is not known whether TPP binds to the alpha or the beta subunit. A putative TPP binding motif as reported by Hawkins et al. (12) for TPP-dependent enzymes is found only in the E1alpha subunits of the alpha-keto acid dehydrogenase complexes. Based on the sequence similarities between E1beta subunits of branched chain keto acid dehydrogenase and transketolase from several species, Zhao et al. (30) have recently proposed that the E1beta subunit may participate in TPP binding. X-ray crystallography studies of transketolase from S. cerevisiae have demonstrated that the TPP binding site is located in a deep cleft between the two subunits (homodimer), and residues(187-198) from both subunits interact with TPP(31) . X-ray crystallographic studies also revealed that the TPP binding site in transketolase is lined with several conserved amino acids such as His, Asp, Arg, Leu, and Ile(32) . However, the identification and exact role of specific amino acid residue(s) in TPP binding of TPP-dependent enzymes is still unclear. This paper is the first evidence for the localization of a specific tryptophan residue in the E1beta subunit at or near the TPP binding site in the TPP-dependent mammalian E1.


FOOTNOTES

*
This work was supported by U. S. Public Health Service Grant DK20478 (to M. S. P.) and DK18320 (to T. E. R.) and by Kansas Agriculture Experimental Station Contribution 93-303-J (to T. E. R.). 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. Tel.: 716-829-3074; Fax: 716-829-2727.

(^1)
The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase; TPP, thiamin pyrophosphate; NBS, N-bromosuccinimide; HPLC, high performance liquid chromatography; MCD, magnetic circular dichroism.


ACKNOWLEDGEMENTS

We are thankful to Drs. Ganesh K. Kumar, Lioubov Korotchkina, and Joyce E. Jentoft of Case Western Reserve University for helpful discussions and critical reading of the manuscript. We are indebted to Gary A. Radke for preparation of bovine pyruvate dehydrogenase complex and resolution of this complex.


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