Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) – evidence for tertiary and quaternary interactions

Review

Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylasesby substrate and thiamin diphosphate.Mg(II) ^ evidence for tertiary and

quaternary interactions

Frank Jordan a;*, Natalia Nemeria a, Fusheng Guo a, Irina Baburina a, Yuhong Gao a,Ara Kahyaoglu a, Haijuan Li a, Jue Wang a, Jizu Yi a, John R. Guest b,

William Furey c

a Department of Chemistry, Rutgers, the State University, Newark, NJ 07102, USAb Department of Molecular Biology and Biotechnology, University of She¤eld, She¤eld S10 2TN, UK

c Department of Biocrystallography, VA Hospital, Pittsburgh, PA, USA

Received 2 December 1997; revised 20 February 1998; accepted 9 March 1998

Abstract

The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvicacid with C221 located on the L domain at s 20 Aî from the thiamin diphosphate (ThDP). To trace the putative informationtransfer pathway, substitutions were made at H92 on the K domain, across the domain divide from C221, at E91, next to H92and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutionswere made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kineticparameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91,E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway forinformation transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvatedehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands arevery important to activation of the enzymes by cofactors. ß 1998 Elsevier Science B.V. All rights reserved.

Keywords: Pyruvate decarboxylase; Pyruvate dehydrogenase; Pyruvate; Thiamin diphosphate.Mg(II) ; Acetyl CoA; (Yeast) ; (E. coli)

0167-4838 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 0 7 5 - 2

Abbreviations: ThDP, thiamin diphosphate; PDC, pyruvate decarboxylase (EC 4.1.1.1) ; scpdc1, wild-type pyruvate decarboxylaseisolated from Saccharomyces cerevisiae ; WT, wild-type PDC, the PDC variants have the same sequence as the scpdc1 except for thesubstitutions indicated, the pre¢x letter is the amino acid in the WT PDC at the position indicated, the su¤x letter is the result ofsubstitution; PDHc, the Escherichia coli pyruvate dehydrogenase multienzyme complex, consisting of E1, E2 and E3 proteins; 3-lipPDHc, overexpressed PDHc with three lipoyl domains in E2; 1-lip PDHc, PDHc with a single lipoyl domain in E2; parental E1, the E1produced from the pGS878 plasmid; the E1 variants are produced from the DNA encoding the parental E1; CPB, (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid; nH, the Hill coe¤cient

* Corresponding author. Fax. +1 (973) 353-1264; E-mail : [email protected]

BBAPRO 35657 29-6-98 ^ Pagina 287 Cyaan Magenta Geel Zwart

Biochimica et Biophysica Acta 1385 (1998) 287^306

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2882.1. Bacterial strains and plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2892.2. Puri¢cation of the 1-lip, 3-lip PDH complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2892.3. Resolution of the components from the PDHc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2892.4. Activity measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2892.5. Fluorescence studies on ThDP binding to E1 and variants of E1 . . . . . . . . . . . . . . . . 2892.6. Kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2903.1. Substrate activation of yeast pyruvate decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . 2903.2. Activation of PDHc from E. coli by ThDP and pyruvic acid . . . . . . . . . . . . . . . . . . . 299

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

1. Introduction

The insightful alignment of the sequences of thia-min diphosphate (ThDP)-dependent enzymes [1], fol-lowed by reports on the high resolution 3-D struc-ture of three such enzymes [2^5], has enabled manygroups to undertake detailed structure-function cor-relation studies of several of the key enzymes thatutilize ThDP.

The authors have been interested in the delineationof the regulation of two such enzymes, pyruvate de-carboxylase (PDC) from Saccharomyces cerevisiaeand the pyruvate dehydrogenase multienzyme com-plex (PDHc) from Escherichia coli at an atomic leveldetail. There are multiple reasons for selecting thesetwo enzymes. PDC from yeast is subject to substrateactivation, as well as to activation by ThDP.Mg(II).However, ThDP is tightly bound at the pH optimumof PDC (6.0), and is only released above pH 8^8.5.PDHc, on the other hand, has a pH optimum at 7^7.5, and the binding of ThDP appears to be rapidlyreversible, enabling a number of groups, most nota-bly that of Bisswanger [6,7], to establish that theenzyme is activated by its cofactor. Herein is pre-sented a summary of the progress made in the au-thors' labs concerning the identi¢cation of amino

acids that participate in substrate activation and cat-alytic mechanism of PDC, studies guided by the re-sults of the X-ray structure. We also present evidence(based on site-directed mutagenesis only, as there isno X-ray structure of E1 to date) on the activation ofE. coli PDHc by ThDP.Mg(II) and substrate andsome of the residues that could be identi¢ed to par-ticipate in these events, as well as some that havebeen ruled out.

2. Materials and methods

Pyruvate decarboxylase was overexpressed in E.coli as described elsewhere [8]. Directed mutagenesisprotocols used PCR technology and these will bepublished elsewhere. The enzyme was puri¢ed ac-cording Farrenkopf and Jordan [9].

PDH complex was overexpressed in both a singlelipoyl (1-lip PDHc) and triple lipoyl domain con-struct (3-lip PDHc), and all of the substitutions re-ferred to in this contribution were made in the E1component of the 1-lip PDHc construct [10]. ThepGS878 plasmid encoding the DNA sequence forE1 was transformed into E. coli TG1 strain, andsite-directed mutagenesis was carried out on this


F. Jordan et al. / Biochimica et Biophysica Acta 1385 (1998) 287^306288

plasmid using the Quick Change site-directed muta-genesis kit from Stratagene using the double-stranded pGS878 DNA, two synthetic mutagenic pri-mers complementary to opposite strands of the DNAand reagents supplied with the kit for 16 cycles.

2.1. Bacterial strains and plasmids

E. coli strain JRG 1342 carrying plasmids en-coding 3-lip PDHc (pGS523), 1-lip PDHc(pGS501), and E1 (pGS878) were from the laborato-ries of J.R.G.

2.2. Puri¢cation of the 1-lip, 3-lip PDH complexes

Cultures were grown in the LB medium containingglucose (0.2%) and ampicillin (50 Wg/ml) at 37³C.Expression of the PDH complexes was induced bythe addition of IPTG to 60 WM. The conditions foroverproduction of the 3-lip and 1-lip PDH complexeswere the same as described elsewhere [10,11]. Cellswere disrupted by ultrasonic treatment after preincu-bation with lysozyme (0.6 mg/ml ¢nal concentration)for 15 min. The PDHc was sedimented by ultracen-trifugation (100 000Ug for 3 h) and further puri¢edby chromatography on a Sephacryl S-400 HR col-umn. Fractions containing PDHc were pooled andtreated with ammonium sulfate to 70% saturation.The enzyme was dissolved in bu¡er A (20 mM po-tassium phosphate, pH 7.8, 2 mM Na2EDTA, 1 mMPMSF, and 1 mM benzamidine.HCl) then dialyzedagainst the same bu¡er. The PDH complexes werestored in bu¡er A at 320³C.

2.3. Resolution of the components from the PDHc

E1 was resolved from the parental 3-lip and 1-lipPDH complexes using a thiol-Sepharose 4B a¤nitycolumn (2.5U20 cm) under conditions described forresolution of E1 from PDHc isolated from Azoto-bacter vinelandii [12]. The E2-E3 subcomplex wasresolved from 3-lip PDHc on a Sepharose CL-6B(1.5U100 cm) column under alkaline conditions.PDHc was dialyzed against 50 mM Tris bu¡er, pH9.0, containing 0.1 mM benzamidine-HCl, 0.15 MNaCl, 0.1 mM PMSF for 2 h, before applying to acolumn equilibrated and eluted with the same bu¡er.Analysis by SDS-PAGE revealed that the ¢rst peak

contains the E1 component and a trace amount ofE2-E3 subcomplex, while the second peak containsE2-E3 subcomplex.

Overexpression and puri¢cation of the E1 pro-duced from the pGS878 plasmid (called the parentalE1) and the cysteine variants of this protein will bepublished elsewhere [40].

2.4. Activity measurements

The overall enzymatic reaction of the PDH com-plexes was assayed using a Varian DMS 300 spectro-photometer or a Cobas Bio (Roche Diagnostics,Somerville, NJ) automated centrifugal analyzer,monitoring the pyruvate-dependent reduction ofNAD� at 340 nm. The incubation medium containedin 1 ml (DMS 300) or 0.25 ml (Cobas-Bio) test vol-ume: 0.1 M Tris-HCl, pH 8.0; 1 mM magnesiumchloride; 2 mM sodium pyruvate; 2.5 mM NAD� ;0.13^0.2 mM coenzyme A; 0.2 mM ThDP; and 2.6mM DTT at 27³C. One unit of activity is de¢ned asthe amount of NADH produced (Wmol) per min permg protein.

The activity of the E1 component resolved from 1-lip PDHc or of parental E1 was measured after re-constitution with an excess of the E2-E3 subcomplexusing the NADH assay for PDHc activity. E1 activ-ity was also assayed according to a mechanism-basedE1-speci¢c assay developed at Rutgers [13].

2.5. Fluorescence studies on ThDP binding to E1 andvariants of E1

Fluorescence measurements were performed at25³C with the SLM8100 spectro£uorimeter. The ex-citation wavelength was 295 nm and the emissionspectrum was recorded in the 300^450 nm range in1 ml or 3 ml quartz cuvettes. The concentration ofparental E1 or variants of E1 with the C259N,N258Q, G231A, G231S substitutions was 0.06 mg/ml in 20 mM KH2PO4 bu¡er (pH 7.5). The excita-tion and emission monochromator slit width were4 nm. The KaleidaGraph V.3.0 software was usedfor data processing. The emission maximum for tryp-tophan at 335 nm was determined by ¢tting thepoints from the peak using the KaleidaGraph com-puter program. The £uorescence quenching datawere analyzed using the equations below:


F. Jordan et al. / Biochimica et Biophysica Acta 1385 (1998) 287^306 289

�13F=Fo� � ff aW�Q�=�Kd � �Q��g �1�

where Fo is the intrinsic £uorescence of E1 in theabsence of quencher; F is the observed £uorescenceat concentration of quencher [Q], fa is the fractionaldegree of £uorescence at concentration of quencher[Q], Kd is the dissociation constant;

Fo=�Fo3F� � f1=�f aWK svW�Q��g � �1=f a� �2�

the Lehrer equation, where Ksv is the Stern-Vollmercollisional quenching constant; and

�vF=FoU100� � f�vFmax=FoU100�U�Q�g

=�Kd � �Q�� 3�

where (vF/FoU100) is the percent quenching (per-cent change in £uorescence relative to the initial val-ue) following addition of ThDP.

2.6. Kinetic analysis

To interpret the data on PDC, two di¡erent sets ofanalyses were carried out. The conventional analysisis presented as Vmax (or kcat), Vmax/S0:5 (since theenzymes are activated, this is a better measure thanKmP), the Hill coe¤cient nH ; while an alternativeanalysis due to Alvarez and Schowen [14] assumesthat there are two substrate binding sites, and col-lects terms as Vmax (or kcat), Vmax/A and Vmax/B, acollection of constants zero, second and ¢rst order insubstrate. The analysis is further complicated by thepresence of very strong inhibition at high substrateconcentrations, implying that yet a third substratemolecule may also intervene.

3. Results and discussion

3.1. Substrate activation of yeast pyruvatedecarboxylase

3.1.1. Background and kinetic evidenceIt had been reported by a number of groups, in-

cluding Boiteux and Hess [15] and Schellenbergerand Hu«bner and coworkers [16,17], that PDC fromyeast gives sigmoidal vo vs. [pyruvate] plots. It was

found that pyruvamide, a substrate surrogate thatcannot be decarboxylated, can also activate thePDC ([16]; at least 40 mM pyruvamide is requiredfor saturation). Schellenberger and coworkers alsogathered considerable evidence to show that a cys-teine residue of PDC is the site of substrate activa-tion, and it was further suggested that the py-ruvate may form a hemithioketal adduct with thecysteine. Schowen and coworkers in recent pu-blications based on transition state analysis sug-gested that PDC is activated with the substrate o¡the cysteine, opening the entry to the active site,and unactivated when the hemithioketal adduct isformed and closes the entrance to the active center[18,19]. Such a mechanism may require addition-elimination of substrate at the regulatory site witheach turnover.

3.1.2. E¡ect of activator on rates and regiospeci¢cityof enamine/C2K-carbanion protonation

In Newark, we have studied for many years theinteraction of PDC with compounds with the struc-ture X^C6H4^CHNCHCOCOOH and showed themto be mechanism-based inactivators [20]. On decar-boxylation by PDC, such compounds are convertedto an enamine/C2K-carbanion that is a visible chro-mophore [21]. The enamine can be protonated eitherK or Q to the thiazolium ring, leading to not onlycinnamaldehydes, but also to dihydrocinnamic acids[22]. The molar ratio of dihydrocinnamic acid tocinnamaldehyde varies with the substituent X, andis as low as 40:1 for the p-£uoro substituent, andis 3:1 in the absence but 2:3 in the presence of sat-urating pyruvamide for unsubstituted phenyl rings.This evidence suggested that protonation of the en-amine is under control of the activation mechanism[22]. The rate of formation of the enamine could bemeasured directly and was found to be in£uenced bysubstrate activation, since saturating pyruvamide in-creased the rate of enamine formation by a factor ofas much as 60 [23]. Importantly, enamine formationin the presence of pyruvamide could be shown to bekinetically competent with the turnover number forpyruvic acid.

3.1.3. Identi¢cation of cysteine 221 as the substrateactivation trigger

We have carried out a series of studies designed to



identify the site at which substrate activation is trig-gered, and the pathway along which the informationis transmitted to the active center. It was ¢rst foundthat the protein resulting from the spontaneous fu-sion of the pdc1 and pdc6 genes (the protein retainedamino acids 1^45 from pdc1 and amino acids 45^563from pdc6) possessed a very useful property so far ascysteines are concerned, namely it had a single cys-teine at position 221, while pdc1 possesses four, atpositions 69, 152, 221 and 222. It could be demon-strated that the presence of Cys221 was su¤cient forsubstrate activation [24]. Next, mutant yeasts withthe C221S, C222S single and the C221S/C222Sdouble substitutions were prepared then transferredinto a high expression vector in E. coli. The PDCswith these substitutions were puri¢ed to homogeneityand were all active. It was demonstrated thatcysteine at position 221, but not at position 222,is required for substrate activation [8,25]. It is im-portant to note that in the C221S variant (and inthe C221A variant): (a) there is a change from sig-moidal to hyperbolic kinetics for pyruvate; (b) thelag period in product formation is abolished; (c)pyruvamide no longer has any e¡ects; but, (d) thereis still 20^30% activity remaining compared to thewild-type (WT) PDC. Therefore, activation is clearlynot an all-or-none phenomenon. Surprisingly, inview of the 3-D structure of PDC from bothSaccharomyces uvarum [4] and Saccharomyces cerevi-siae [5], C221 is more than 20 Aî from ThDP, and islocated on the L domain, while ThDP is located be-tween the K and Q domains, also at a subunit inter-face.

3.1.4. Protein chemical evidence for reactivityat C221

Dikdan at Rutgers [26,27] used radioactive ThDPand p-ClC6H4^CHNCHCOCOOH ([1-14C]CPB and[3-3H]CPB) to show that concomitant with inactiva-tion, the cinnamaldehyde product p-ClC6H4^CHNCHCHO resulting from the decarboxylationof CPB, but not the CPB itself, becomes covalentlybound at C221 (there was 3H, but no 14C detected inthe peptide encompassing C221 and C222). This pro-vided the ¢rst direct demonstration of the highly re-active character of C221 as a nucleophile, other thanits reactivity towards Hg derivatives noted during theX-ray studies.

3.1.5. The state of ionization of cysteines, includingC221, on PDC at the pH optimum

We used FT-IR spectroscopy to show that three ofthe four cysteines on PDC are ionized at pH 6.0 [28].The approach was inspired by the observation thaton hemoglobin one can observe S-H stretching vibra-tions near 2550^2560 cm31, an IR window devoid ofany other vibrational bands. FT-IR experimentswere conducted on PDC and several variants atand near pH 6.0, the pH optimum of the enzyme.PDC has a very narrow pH activity range betweenpH 5 and 7, where both Vmax and Vmax/S0:5 exhibitpH-dependent changes. The FT-IR spectrum of theC221S, C222S, C221S/C222S, H92A, C152A variantsalong with that of the WT PDC was examined. All,except the C152A variant, possessed an S^H bandwith the same magnitude and frequency, while theC152A variant showed no sign of an S^H band with-in experimental error. The areas under the bands at2558 cm31 were compared to protein bands at 1455cm31 present in all samples, allowing normalizationof the results. The width of the band suggested thatthe single S^H is located in a hydrogen bonded en-vironment with an intermediate strength hydrogenbond. Addition of ketomalonate, a reagent that isdirected to C221 [29] had no e¡ect on the IR bandin question, further con¢rming the absence of the S^H at C221 in WT.

To complement the FT-IR results, isoelectric fo-cusing experiments were undertaken on the same var-iants. Our working hypothesis was that for an undis-sociated S^H group, substitution by Ser^OH willlead to no changes in the isoelectric point, whereasif the cysteine is ionized at the same pH, its replace-ment by Ser^OH will change the isoelectric point.The experimentally determined isoelectric pointsare: 5.61, 5.70, 5.66, 5.84, and 5.51 for the WTand the C221S, C222S, C221S/C222S and H92APDC variants, respectively. The data indicate thatthe isoelectric points of the cysteine variants all shiftcompared to that of WT PDC, in the direction ap-propriate for removal of negative charges. The shiftsare ca. 0.09^0.10 units per integral charge. The H92Asubstitution leads to a shift that suggests that H92exists in the imidazolium form at pH 6.0.

Using the DELPHI program from Biosym, an at-tempt was made to trace the origin of the electro-static ¢eld that is responsible for the dramatic pKa



perturbations suggested by the results (the pKas ofC69, C221 and C222 are deduced to be lower than5.6, compared with the aqueous value near 8.5). Suf-¢cient positively charged residues are present to ac-count for the diminished pKa of C221 and C222.Substitution of these positively charged amino acidsby neutral ones predicted a pKa increase at the cys-teines, the magnitude of the calculated increase ofcourse depends on the assumed dielectric constant.An inspection of the X-ray structure indicates thatthere is a backbone carbonyl oxygen from aminoacid E148 that is 3.3 Aî from the sulfur of C152,poised to participate in a hydrogen bond with theC152 S^H group. This interaction may explain whythis cysteine alone may have a normal pKa, not assuppressed as that of the other three cysteines.

Based on results from this laboratory, it appearsthat C221 is ionized at pH 6.0. The pK perturbationis probably the result of the positive charges that

surround C221. It is also evident from model build-ing based on the X-ray atomic coordinates that oncepyruvate is bound to C221 on the L domain, forexample via a hemithioketal adduct, the bound sub-strate would be within van der Waals contact of theH92 imidazole side chain located on the K domain.This side chain appears to be in the imidazoliumionization state at pH 6.0 according to IEF results.A signi¢cant role of the PDC protein is to create astrong positive ¢eld thereby enhancing the nucleophi-licity of the C221 sulfur, since thiolate is a muchstronger nucleophile than the thiol conjugate acidform. This motif is reminiscent of other proteinscharged with enhancing the nucleophilicities of cys-teines, such as papain, glyceraldehyde-3-phosphatedehydrogenase and thiolsubtilisin (a serine proteasein which the active center serine is replaced by acysteine), all of which are believed to operate withan imidazolium-thiolate ion pair at their reactive cen-

Fig. 1. The pathway on pyruvate decarboxylase from C221 to ThDP.



ters. While formation of a hemithioketal between thepyruvate and a regulatory cysteine was already sug-gested by Schellenberger and Hu«bner, the evidencehere presented make such predictions less specula-tive, yet, no direct evidence exists to date for theformation of such an adduct.

3.1.6. On the trail of the activation pathway

3.1.6.1. Variants at position H92. According to amodel of pyruvate bound at or near C221, H92should be the ¢rst residue a¡ected by addition ofpyruvate to C221, being forced to move slightly(see Fig. 1 for the relationship of residues). H92was substituted [29] for A, G and K to test: (a)whether H92 participates in the substrate activationpathway; and (b) what kinds of interactions occurbetween pyruvate bound to C221 and H92. TheH92C/C222S variant was also prepared in an attemptto convert PDC to a permanently activated form ofthe enzyme.

The H92A and H92G substitutions reduce the Hillcoe¤cient from 2 to 1.2^1.3. With the H92K substi-tution the positive charge is conserved and a Hillcoe¤cient of 1.5^1.6 results, intermediate betweenthat for the WT PDC (2.0) and the H92G variant(1.2), suggesting that the positive charge at H92 isimportant. During the work with the H92 variants itwas noticed that the H92A, H92G and H92C/C222Svariants are very unstable under the usual storageconditions: these proteins lost some of their activityand cooperativity within two weeks. However, the

kinetic parameters of the H92K variant remainedstable for several months, this variant retains its pos-itive cooperativity. The results provide clear indica-tion of the importance of a positive charge at posi-tion 92 in the signal transmission between domains,and in helping to stabilize the protein, in addition tomaintaining positive cooperativity. Lysine wouldlikely be protonated in the entire pH range of interestand the kcat vs. pH pro¢le for the H92K variant doesshow di¡erences from the WT PDC.

The pH dependence of steady-state kinetic param-eters was determined for WT PDC and the C221S,C222S, C221S/C222S, H92G and H92K variants.Perhaps most striking is the behavior of the Hill co-e¤cients. While the Hill coe¤cients for WT PDC andthe C222S variant are pH-dependent and bell-shapedwith the maximum at VpH 6.0, those for the C221S,H92G and H92K variants are nearly pH-independent.A comparison of the Hill coe¤cients of the H92Aand H92G variants with those of WT PDC suggeststhat the length of the side chain at position 92 maybe important, while a comparison with the H92Kvariant shows that the positive charge at position92 can compensate, in part, for the loss of the histi-dine. Based on the pH dependence of the Hill coef-¢cient in WT PDC and its pH independence in theC221S, H92G and H92K variant PDCs, and the¢nding that C221 is dissociated at pH 6.0 (i.e. itspKa is lower than 6; see [27]), we suggest that thepKas apparent from the pH dependence of the Hillcoe¤cient in the WT PDC pertain to C221 (V5.2)and H92 (V6.4) [29].

Scheme 1.



The pH dependence of the steady-state kinetic pa-rameters provides considerable insight to the sub-strate activation pathway: in the WT PDC, there isclear evidence of both V and V/S0:5 being modulatedby at least two ionizable groups according to thebell-shaped pro¢les. In the Dixon-Webb formalism,these would pertain to protonic equilibria involvingthe EWS complex and free E, respectively. The Vmax/S0:5-pH plots have virtually the same shape as theVmax/A-pH plots. According to the mechanism de-rived by Alvarez et al. [14], Vmax/A re£ects on theenergetics of transition states starting with the addi-tion of the ¢rst pyruvate to PDC (presumably at theregulatory site) and terminating with the ¢rst irrever-sible step, i.e. decarboxylation. The term Vmax/B re-£ects the energetics of transition states starting withthe EWS complex and terminating with decarboxyla-tion. Finally Vmax (or kcat) reports on transition stateenergies for the decarboxylation step (formation ofthe enamine/C2K-carbanion), followed by productrelease (broadly de¢ned), including protonation ofthe enamine and release of acetaldehyde fromThDP (Scheme 1). When comparing the plots ofWT PDC with those for the C221S variant, onecan conclude the following: (a) The kcat-pH pro¢leshave similar shapes, but the pKas are somewhat clos-er to each other in the variant than in the WT PDC.There is some perturbation in the pKas of the activecenter residues. (b) A larger change is sensed in theV/A(or kcat/S0:5)-pH and V/B-pH plots, as expectedfrom the above-mentioned dramatic changes in theHill coe¤cients. Evidently, an ionizable group with apKain the acidic region is being perturbed by thesubstitution.

The shape of the kcat-pH, kcat/S0:5-pH (or V/A-pHand V/B-pH) plots for the H92G variant are verysimilar to those for the WT PDC, signaling thatthere was no major perturbation in the rate-limitingsteps. There is one notable di¡erence. The pH opti-ma of the kcat/S0:5-pH (or V/A-pH) plots and thesingle pKapp in the V/B-pH plot have experienced ashift to the acidic region by perhaps as much as 0.5units. This provides further evidence that the bindingof pyruvate to the regulatory site can sense thechanges in ionization state at position 92.

The results on the H92K variant were surprising.The kcat does not decrease with increasing acidity to atleast pH 5.0, in contrast to the other variants studied.

This result provides striking con¢rmation for the par-ticipation of H92 in the transmission of signal to theactive center, since kcat re£ects energetics of transitionstates in decarboxylation and product release. Aplausible explanation of the observation is that thegroup(s) responsible for the acid limb in the kcat-pHpro¢le of WT PDC has (have) experienced enhancedacidity, so that even at pH 5 it is (they are) fullydissociated. In other experiments (Gao, Guo, Wangand Jordan, unpublished), there is evidence accumu-lating that several residues surrounding ThDP (butnot near C221 or H92), such as E51, E477, D28,H114, H115) have an impact on this region of thekcat-pH pro¢le. As can be seen in Fig. 1, the pathwayfrom H92 to E91 to W412 and the 411^415 loopleads to the active center and involves residues thatare intimately involved in ThDP binding. Further-more, these residues are in close proximity to theactive center acid-base groups mentioned above.

While the decrease in regulatory properties ob-served with substitutions at C221 and H92 mightbe due to a distortion of the tertiary structure result-ing from amino acid substitution, the observationthat these substitutions have their most dramatic ef-fects on cooperativity rather than activity suggeststhat changes in the tertiary structure of the enzymeare not signi¢cant but subtle.

3.1.6.2. Attempts to convert PDC to a permanentlyactivated form. With the assumption that there is ahemithioketal adduct formed between the substrateand C221, we used three di¡erent alkylating agentsto install a mimic. An H92C/C222S double mutantwas created with the expectation that it may react atboth the C92 and C221 positions with 1,3-dibromo-acetone. Indeed, addition of 0.1 mM 1,3-dibromo-acetone to this double mutant, but not to the WTPDC, reduced the Hill coe¤cient to 1.0, while onlyreducing the enzyme activity by 30%. As a control, ittook more than 5 mM 1-bromo-2-butanone toachieve the same reduction in nH. Also, addition of1,3-dibromoacetone or 1-bromo-2-butanone had thesame e¡ect on the WT PDC. In contrast, it required30 mM iodoacetate to reduce the n of WT PDC to1.14. A plausible explanation of all of these results isthat all three reagents alkylated C221, but their ef-fects vary considerably. Based on the kinetic evi-dence, it is likely that 1,3-dibromoacetone cross-



linked the H92C/C222S variant. While addition ofpyruvamide converts the enzyme to an activatedform, as does pyruvate, some of the other reagents,such as iodoacetate may simply be destroying theallosteric interactions.

Lobell and Crout [30] reported modeling studiessuggesting that pyruvate can form two hydrogenbonds, to the NH and CO of S311, a residue nearC221. These bonds could hold pyruvate in positionfor the formation of a hemithioketal. Substitution of

pyruvate by pyruvamide allows the same interac-tions, but carboxymethylation of C221 would not.Results with iodoacetate, 1,3-dibromoacetone and1-bromo-2-butanone tend to support the importanceof a recognition site for the keto group: (a) iodoace-tate has a much lower reactivity at the regulatory sitethan the two bromoketones; (b) reaction of theH92C/C222S variant with the two bromoketonesleads to improved S0:5, whereas all such alkylationof the WT PDC increases S0:5. The bromoketonesintroduce the keto function that apparently convertsthe variant to its activated form (n tends to 1.0, andS0:5 decreases with added bromoketone). Unfortu-nately, the C221 and H92C variants tend to be dena-tured with time, hence there is no structural con¢r-mation of this hypothesis to date.

Fig. 2. Active center environment in pyruvate decarboxylase.



3.1.6.3. Variants at position E91. As was sug-gested elsewhere [5], any distortion sensed by H92on binding of pyruvate at C221 may also move thebackbone leading to E91. Just as the informationfrom C221 is transmitted to H92 on a di¡erent do-main, the information from E91 on the K domain istransmitted via a hydrogen bond to the backboneNH of W412 on the Q domain (the three domainseach comprise circa 185 amino acids), linking up tothe 411^415 loop that envelopes the pyrimidine ring,providing not only two of three conserved hydrogenbonds (I415 to N3P and G413 to N4P), but also thehydrophobic side chain occupying the `V' conforma-tional pivot (I415, see below). To test for the partic-ipation of E91 in the information transfer pathway,the E91Q, E91D and E91A variants were prepared(H. Li, unpublished). The speci¢c activities (units/mgin parentheses) diminished in the order WT PDC(60)sE91Q (16)sE91D (11)EE91A (2). This isthe same order as was found for the stability of thevariants, measured from the temperature dependentactivity decrease pro¢le. Perhaps most interestingly,the Hill coe¤cients are pH-dependent and give rise

to similar shapes for WT and E91D variant PDCs,but are pH-independent and much diminished forthe E91A (1.4) and E91Q (1.1) respectively. We con-clude that the interaction of E91 with the backboneNH of W412 has two components: the length of theside chain and its hydrogen bonding ability ensurestability, and the negative charge ensures the activa-tion.

3.1.7. Amino acids at the active centerSeveral residues likely to be involved in catalysis

and/or binding have been identi¢ed (Fig. 2).

3.1.7.1. Isoleucine 415. One of the striking ¢nd-ings from the X-ray structures of PDC, pyruvateoxidase from Lactobacillus plantarum (POX) andyeast transketolase (TK) is the presence of the Vcoenzyme conformation with respect to the disposi-tion of the two aromatic rings attached to the bridg-ing methylene group. This is in contrast to the F (forfree) and S (for substituted at the reactive C2 atom)conformers preponderant for thiamin and its deriva-tives. The conformers are usually de¢ned in terms of

Table 1E¡ect of substitutions near the substrate activator and ThDP sites on kcat and Hill coe¤cients at pH 6.0, 25³C

ThDP site Activation pathway

PDC variant Relative kcat n PDC variant Relative kcat n

WT 1.0 2.0Asp28Ala 0.015 1.6 Cys221Ala 0.3 1.0Asp28Asn 0.0079 1.7 Cys222Ser 0.8 1.9

Glu51Gln 0.016 1.1 Cys152Alaa 1.0 2.0Glu51Asp 0.13 1.6Glu51Asn 0.011 1.1 His92Gly 0.5 1.2Glu51Ala 0.010 1.1 His92Lys 0.5 1.7

Ile415Val 0.081 1.5 Glu91Ala 0.039 1.4Ile415Thr 0.035 1.6 Glu91Gln 0.27 1.1Ile415Ala 0.023 1.9 Glu91Asp 0.23 1.9

Glu477Asp 0.0096 2.0Glu477Gln 0.0075 2.0Glu477Asn 0.0165 2.1

His114Phe 0.011 2.0His115Phe 0.014 2.1His114Phe/His115Phe 0.0085 1.1aC152 is the third exposed cysteine.



dihedral angles for rotation around the C5P^Cbridge

and Cbridge^N3 atoms, called xP and xT, respec-tively. Early Lennard-Jones type calculations sup-ported these experimental ¢ndings [31,32]. Therewere, however, a few instances of V conformers iden-ti¢ed in structures where the 4P-amino group wasmissing. The principal attribute of the V conformeris that it brings the N4P and C2 atoms into closeproximity. Both Schellenberger's group and theRutgers group have speculated on the importanceof this proximity, i.e. the possible participation ofthe amino group in catalysis (see recent discussionin [5]). On all enzymes examined by X-ray, the Vconformation is supported by a large hydrophobicamino acid side chain: I415 on PDC, Met on POXand Leu on TK. In view of I415 being on the puta-tive substrate activation pathway emanating fromC221 and terminating at the active center, and onaccount of the (so far) universality of the V confor-mation observed for enzyme-bound ThDP, a system-atic study was undertaken by varying the size of theside chain at position 415 in PDC from Saccharomy-ces cerevisiae. So far, the I415L, I415V, I415T,I415A, I415S and the I415M variant PDCs were pre-pared (Guo, Zhang, Kahyaoglu, Farid and Jordan,in press). Brie£y, a diminution in the size of thegroup at position 415 leads to diminished speci¢cactivity, especially re£ected in their kcat values com-pared to that of WT PDC, suggesting that I415 has arole in the stabilization of the transition state fordecarboxylation and/or product release. Details ofthese studies will be presented elsewhere, here weonly note the e¡ect of these substitutions on sub-strate activation. The nH values in Table 1 indicatethat the cooperativity is not dramatically a¡ected bythese substitutions.

3.1.7.2. Glutamate 51. This amino acid is posi-tioned opposite the N1P pyrimidine atom, and a car-boxylic acid appears to be highly conserved at thisposition in all of the X-ray structures reported todate. We had hypothesized that this residue wouldhelp to maintain the 4P-imino tautomeric form of thepyrimidine, so that it can participate in intramolecu-lar proton abstraction from the thiazolium C2 posi-tion. The E51D, E51Q, E51N and E51A variantswere studied (Gao and Wang, unpublished). The spe-ci¢c activities of each were substantially smaller than

of the WT PDC, clearly signaling that this is animportant residue. Extensive steady-state kineticstudies were carried out in the entire pH range ofactivity on all variants. Most relevant to this discus-sion, the Hill coe¤cient changed: in the WT nH ispH-dependent with a maximum of 2.0 at the pHoptimum of 6.0 [29]; in E51D nH is pH-dependentwith a range of 1.5^2.2 and a minimum at pH 6.0; inE51Q nH is pH-dependent with a range of 1.0^1.8and a minimum at pH 6.0; in E51N nH is pH-inde-pendent with a value of 1.2^1.3 and ¢nally in E51AnH is pH-independent with a value of 1.0^1.3. Theresults provide dramatic evidence for the importanceof the charge in the substrate activation pathway atC221, H92, E91 and E51. Results for the E51Q andE51A variants were reported at pH 6.0 [34,35]. TheE51 variants are of very low activity (as already ob-served on transketolase in [33]).

3.1.7.3. Aspartate 28, histidines 114 and 115, gluta-mate 477. These amino acids are within easy reachof not only the locus of activity (the N4P and C2atoms), but also the C2K atom and carboxyl groupof the coenzyme-bound pyruvic acid. For this reason,they are also obvious candidates for substitution.That E477 has an important catalytic role was sug-gested in a computational study of the principalPDC catalyzed reactions [30].

We have prepared to date the E477Q, E477D,E477N, D28A, D28N, H114F, H115F and H114F/H115F variants (Guo and Wang, unpublished). Asindicated in Table 1, all of them had diminished ac-tivities. Single substitutions at any of these positionshad virtually no impact on the Hill coe¤cient. Re-markably, the H114F/H115F doubly substituted var-iant had a speci¢c activity not very much below theH114F and H115F singly substituted ones, yet, themodi¢ed enzyme was no longer subject to substrateactivation. An inspection of the model of the enzymeshows that these side chains abut the W412 indoleside chain, the HNN1 imidazolyl atom of H115 ishydrogen bonded to the CNO of W412.

3.1.8. Current working hypothesis on regulation ofand catalysis by PDC

The picture that emerges to date is based in parton the X-ray structure and in part on the studies hererelated. An inspection of a space-¢lling model of



PDC indicates that C221 and ThDP are the onlyloci that are readily accessible from the surface ofPDC. In a recent publication, there was substantialevidence presented that these two sites constitute thesole reactive sites on the enzyme [27]. The ¢rst eventin the reaction is the docking of substrate at C221,we believe covalently, although no evidence is yetavailable except for the reaction with the cinnamal-dehyde (see above). Docking of pyruvate at C221 onthe L domain displaces H92 on the K domain by acombination of charge and van der Waals interac-tions, in turn shifting the main chain, includingE91. This movement then impacts on the interactionof E91 with the main chain NH of W412 on the Qdomain. W412 in turn is hydrogen bonded using itsmain chain carbonyl oxygen to H115, and movementof the main chain impacts on the position of G413,whose carbonyl oxygen provides the sole hydrogenbond to the N4PH of the aminopyrimidine ring. Sofar, it appears that correct positioning of this hydro-gen bond may indeed be responsible for the actualsubstrate activation event, subtle as it is. The onlyother side chain at the active center whose substitu-tion leads to abolition of homotropic activation isE51. Here again, there are two factors, any substitu-tion very much reduces the activity, but the E51Dvariant still preserves the homotropic activation. The¢rst, most crucial step may indeed be proton transferfrom the thiazolium C2 to the imino N4P atom. Thisrequires the E51, now shown both at Halle [34,35]

and at Rutgers. When there is a negatively chargedresidue present at position 51 (either WT PDC or theE51D variant), the enzyme can be activated by sub-strate at C221. Once E51 is replaced by alanine, asan extreme replacement, the pathway triggered atC221 can no longer intervene. It is possible thatour observations with the H114F/H115F doubly sub-stituted variant are signaling a similar story, i.e. thepresence of either histidine by itself still allows sub-strate activation originating at C221, but substitutionof both histidines damages the substrate activationpathway. That these two adjacent histidines do notconstitute a distinct activation pathway of their owncan be deduced from the fact that these two histi-dines appear to be also present at the correspondingpositions in PDC from Zymomonas mobilis which isnot subject to substrate activation [36]. So far, wehave not identi¢ed an additional pathway leadingfrom C221 to E51, as we appear to have identi-¢ed the C221CH92CE91CW412CG413CN4PHpathway. We point out that this pathway crossesboth domain and subunit interfaces, and there couldindeed be additional pathways from C221 to the ac-tive center. With all of the evidence at our disposal, itis clear, however, that while PDC is substrate acti-vated, and the activation takes place on the very ¢rststep, unactivated or unactivatable PDC is not an in-active enzyme. It is interesting to observe (and per-haps of great signi¢cance) that the interactions iden-ti¢ed among domains and subunits tend to involve at

Table 2Kinetic parameters for ThDP binding to PDH complexes

Complex Substitution Speci¢c activitya (units/mg) Relative activity (%) Km (mM) ndH

3-lip PDHc 8.43 100 0.0026 0.92E1 (PDHc) 0.0043 0.17b 1.3E1 (resolved) 0.0106 0.034 1.01-lip PDHc 6.86 100 0.0028 0.99E1 (PDHc) 0.0017 0.19b 1.21-lip PDHc G231A 0.067 0.97 unsat.c 6 1^ G231S 0.0019 0.03 ^ ^^ N258Q 1.18 17.0 unsat.c 6 1^ C259S 1.85 27.0 unsat.c 6 1^ C259N 3.94 58.0 0.76 1.0aActivity of 3-lip, 1-lip and variant PDH complexes was determined using the NADH assay in the presence of 0.2 mM ThDP. E1 ac-tivity in the 3-lip, 1-lip PDH complexes or of resolved E1 was assayed according to E1-speci¢c assay.bApparent Km.cNot saturated at 5^10 mM ThDP.dHill coe¤cient.



least one charged residue, in some cases two, stronglyimplying that the forces responsible for these inter-actions are electrostatic in origin.

The binding of pyruvate at the C2 thiazolium po-sition may proceed by neutralization of the incipientalkoxide at C2 of pyruvate by the proton from theN4P (the one gained from C2 earlier). The other res-idues around the active center studied at Rutgers sofar have far-reaching e¡ects on Vmax, they have arole in decarboxylation and product release, andprobably lesser roles in the binding of substrate,although a recent report on the Zymomonas mobilisPDC suggested that the double histidines are in-volved in substrate binding [36]. Based on the pH-dependent steady-state kinetic parameters of theseresidues, it appears to be the case that H114, H115,D28 and E477 form a network of residues, a networkthat also involves the water molecule bound at D28.Much remains to be done to gain a better structuralunderstanding of both the regulatory and catalyticmechanism. For now, it appears that we can dissectthe problem by turning o¡ substrate activation atcysteine 221.

3.2. Activation of PDHc from E. coli by ThDP andpyruvic acid

The pyruvate dehydrogenase multienzyme complex(PDHc) is responsible for converting pyruvate to ace-tyl-coenzyme A. PDHc from E. coli is comprised ofthree protein chains, E1, E2 and E3. The E1 compo-nent of PDHc carries out decarboxylation of pyru-vate to ThDP-bound enamine/C2K-carbanion, whilelipoyl-E2 performs subsequent oxidative acetyl trans-fer to produce S-acetyldihydrolipoyl-E2. PDHc, un-like PDC, binds its ThDP less avidly, perhaps due tothe fact that the optimum pH in E1 is shifted tomore alkaline values by 1^1.5 pH units. This attrib-ute of E1 provides a tool for studying the homo-tropic regulation of the E. coli PDHc by its cofactorsThDP.Mg(II) and its substrate pyruvate. We carriedout such studies with complexes containing three (3-lip PDHc) and one lipoyl domains (1-lip PDHc) perE2 chain, and with several variants of the latter,containing substitutions in the putative ThDP foldof E1 (G231A, G231S, C259S, C259N andN258Q). In addition, with a plasmid coding for theE1 only (called the parental E1), we also produced

E1 variants at ¢ve of the six cysteines (the sixth beingavailable from the 1-lip PDHc constructs C259S andC259N).

All of the 1-lip PDHc variants had reduced speci¢cactivities, as reported elsewhere [10,11]. Extensive ki-netic studies were performed in an attempt to deter-

Fig. 3. (A) Typical progress curve for NADH production in theoverall PDHc reaction with 1-lip PDHc at 0.9 WM ThDP con-centration. Inset, Progress curves for NADH production in theoverall reaction with the G231A variant of 1-lip PDHc (see [11]for experimental conditions). (B) ThDP dependence of the lagphase for resolved E1 (see [11] for experimental conditions).



mine the e¡ects of amino acid substitutions on theHill coe¤cients with respect to the ThDP and pyr-uvic acid. All but one of the variants were incapableof being saturated with thiamin diphosphate even atconcentrations s 5 mM (Table 2).

Most signi¢cantly, the striking activation lag phaselasting for many seconds in the parental 3-lip and1-lip PDH complexes (Fig. 3A) and in the resolvedE1 component (Fig. 3B; also attesting to the factthat activation by the coenzyme was localized tothe E1 subunit), was totally eliminated in the vari-ants (see results on the G231A variant in Fig. 3A,inset).

The lag phase at low ThDP concentrations hadbeen extensively studied in the WT enzyme by Biss-wanger and coworkers [6,7]. ThDP-Mg(II) activationappears to originate from the initial interaction ofthe cofactors with the diphosphate-Mg binding site.The stretch of amino acids identi¢ed by Hawkins etal. as the so-called ThDP fold [1], in view of X-raystructural data on related enzymes, is most importantfor creating the appropriate ligand environment forthe Mg(II), that ion in turn forms an inner spherecomplex with the K and L phosphate oxygens ofThDP. The complex dependence of the lag phaseon the ThDP concentration suggests stepwise bindingof the two ThDP.Mg(II) complexes to the E1 dimer.The ¢rst ThDP.Mg(II) is bound with a Kd in themicromolar range, followed by a slow conformation-al equilibration step and leading to a half-saturatedE1 dimer [11]. A single-step binding mechanism wasproposed for the binding of the secondThDP.Mg(II), since we could not resolve kineticallya separate binding step followed by a slow conforma-tional equilibration. When there is no lag phase ob-

served, such as in the variant enzymes with impairedThDP folds, there is a fast, perhaps encounter con-trolled binding step that leads to a very much higherKm in the steady state (at least 100-fold).

3.2.1. Fluorescence studies on the binding of ThDP toresolved E1 (parental E1) and E1 variants withsubstitutions in the ThDP binding fold

The E. coli PDHc has an intrinsic £uorescence,probably due to tryptophan, which diminishes upon

Table 3Quenching of the intrinsic £uorescence of E1 and E1 variants with substitutions in ThDP-binding fold by ThDP in the absence andin the presence of 2 mM pyruvate

Enzyme Substitution Kd (WM)a Kd (WM)b Kd (WM)c

1. E1 ^ 132 þ 19 6.5 þ 0.4 1702. E1 G231S 189.0 2.8 not saturated3. G231A 103.0 10.3 not saturated4. N258Q 130.0 11.8 2605. C259N 99.0 5.3 175aIn the absence of pyruvate.bWith 2 mM pyruvate. The values of Kd obtained at 2^15 WM (parental E1) and 2^40 WM ThDP (E1 variants).cWith 2 mM pyruvate. The values of Kd obtained at 50^400 WM concentrations of ThDP.

Fig. 4. E¡ect of ThDP on the £uorescence emission spectrumof resolved E1. The £uorescence emission spectrum of E1 (0.05mg/ml) in 3 ml of 20 mM KH2PO4 bu¡er (pH 7.5), containing1 mM MgCl2 (curve 1) and the following micromolar concen-trations of ThDP (curve number in parentheses): 4 (2), 8 (3),16 (4), 24 (5), 32 (6), 40 (7), 60 (8), 100 (9), and 200 (10). Insetshows the dependence of the £uorescence decrease (Fo3F) at335 nm on ThDP concentration, where Fo is the £uorescenceintensity of E1 in the absence of ThDP and F is the £uores-cence intensity in the presence of the indicated concentration ofThDP.



reconstitution with ThDP in the presence or in theabsence of pyruvate [37]. We have shown for the ¢rsttime for E1 resolved from E. coli PDHc that onaddition of 4^200 WM concentrations of ThDP, theintrinsic £uorescence of E1 decreased (Fig. 4). Anemission maximum at 335 nm resulted from excita-tion at 295 nm, very likely corresponding to buriedtryptophan residues [38]. The £uorescence quenchingof parental E1 upon addition of ThDP exhibitedsaturation of enzyme sites (Fig. 4, inset) suggestingthat speci¢c changes in interactions resulted. Ananalysis of the data using Eq. 2 revealed that thefraction of the total tryptophan £uorescence (fa)available to quenching by ThDP was 0.695, suggest-ing the participation of only one type of tryptophanresidue with similar exposure and accessibility toThDP. The values of Kd for ThDP calculated withEq. 1 or Eq. 3 are presented in Table 3.

In Table 2 above [11], it was shown that only theC259N-E1 variant of 1-lip PDHc could be saturatedby ThDP (Km = 0.037 mM, compared to 0.0028 mMwith parental 1-lip PDHc). None of the other var-iants could be saturated by even 5^10 mM ThDP.Based on the kinetic evidence, it was concluded thatfor the G231A and G231S E1 variants of 1-lip PDHconly one binding site is saturated by ThDP [11]. Wehere report £uorescence quenching data on theG231A, G231S, C258Q and C259N E1 variants of1-lip PDHc. These substitutions did not change sig-ni¢cantly either the emission maximum at 335 nm orthe £uorescence intensity. The binding curves weresimilar for all variants and the deduced Kds are pre-sented in Table 3, suggest that substitutions in theThDP-binding fold did not induce signi¢cant confor-mational changes in close proximity to the £uoro-phore.

Fig. 5. Quenching of £uorescence of resolved E1 by ThDP in the presence of 2 mM pyruvate. Increasing concentrations of ThDPwere added to 0.06 mg/ml E1 and 1 mM Mg(II) (in 20 mM KH2PO4, pH 7.5 bu¡er) at 25³C and the £uorescence emission spectrumwas recorded in the 300^450 nm range. The emission maximum at 335 nm for di¡erent concentrations of ThDP was determined. (A)Data were treated according to the Lehrer Eq. 2 (Section 2); (B) data presented as a double-reciprocal plot of Eq. 3; (C, D) the con-tinuous line represents the ¢t of the data points at low and high concentrations of ThDP to Eq. 1 using the Kd calculated fromplot B.



3.2.2. Quenching of the E1 £uorescence by ThDP inthe presence of 2 mM pyruvate

According to earlier data, the pyruvate concentra-tion may a¡ect the binding a¤nity of E1 for ThDP[39]. From the kinetic data from this lab [11], the Kd

for ThDP binding was 12.29 WM in the absence and3.73 WM in the presence of 2 mM pyruvate. It wasshown that pyruvate does not change signi¢cantlythe rate constant for ThDP binding, however it de-creases the rate constant of ThDP dissociation [11].Hennig et al. reported Kd for ThDP binding of 16.8WM in the absence of, and 1.4 WM in the presence of2 mM pyruvate as determined by activity measure-ments [37], and 18.1 WM in the absence of, and 1.49WM in the presence of 2.0 mM pyruvate based on£uorescence measurements, suggesting that pyruvateincreases the a¤nity of E1 for ThDP.

The binding of ThDP to E1 resolved from PDHcand to the C259N, N258Q, G231A, and G231S E1variants (resolved from the corresponding PDH com-plexes) was also examined by £uorescence quenchingexperiments in the presence of 2 mM pyruvate. Anal-

ysis of the data according to Eq. 2 or Eq. 3 revealeddownward curvature with two linear regions (Fig.5A,B), in contrast to the straight line obtained inthe absence of pyruvate (not shown). These resultssuggest that the two ThDP.Mg(II) binding sites onthe E1 dimer behave as distinct £uorophores withdi¡erent Kd values, and, alteration of the secondThDP.Mg(II) binding site results once the ¢rst oneis ¢lled. Separate treatment of the data for low (2^40

C

Fig. 7. (A) Michaelis-Menten plot for 1-lip PDHc. The reactionmixture (0.25 ml) in 0.1 M Tris-HCl bu¡er (pH 8.0) contained:2.5 mM NAD�, 2.6 mM DTT, 0.2 mM ThDP, 1 mM Mg(II),0.13 mM CoA. The reaction was initiated by addition of en-zyme (1.6 Wg). Inset, Eadie-Scatchard plot. (B) Michaelis-Men-ten plot for 1-lip PDHc in the presence of 0.65 mM acetyl-CoA. Reaction conditions were the same as in A. Inset, Eadie-Scatchard plot. (C) Dependence of the steady-state rates onpyruvate concentration for the G231A variant of 1-lip PDHc.Reaction conditions were the same as in A. The reaction wasinitiated by addition of enzyme (140 Wg). Inset, Eadie-Scatchardplot.

Fig. 6. Quenching of £uorescence of G231A E1 resolved from the corresponding complex. Reaction conditions were the same as inFig. 5. (A) Data were treated according to Lehrer Eq. 2 (Section 2); (B) data presented as a double-reciprocal plot of Eq. 3; (C) thecontinuous line represents the ¢t of the data points at low concentrations of ThDP to Eq. 1; (D) the saturation curve was not ob-tained at high concentrations of ThDP.



WM) and high concentrations of ThDP (50^400 WM)using a plot of [13(F/Fo)] versus [ThDP] revealedthat both sites were saturable for parental E1 (Fig.5C,D) and for the N258Q and C259N variants of E1(data not presented). However, for the G231A andG231S E1 variants (Fig. 6B,C) saturation was ob-served only at low concentrations of ThDP (2^40WM), but no saturation of the second site was ob-served even at a 50^500 WM ThDP, in accord with

Fig. 8. (A) Michaelis-Menten plot for the C575A variant of pa-rental E1. Reaction conditions were the same as in Fig. 7A.The reaction was initiated by addition of a mixture of E1 (0.7Wg) and E2-E3 subcomplex (4 Wg). Inset, Eadie-Scatchard plot.(B) Michaelis-Menten plot for the C575A variant of parentalE1 in the presence of 0.65 mM acetyl-CoA. Inset, Eadie-Scatch-ard plot.



the kinetic data reported from our labs. The datapresented in Table 2 suggest that in the presence ofsaturating pyruvate concentrations there is a higha¤nity binding site for ThDP with a Kd of 2.8^11.8 WM, and a much lower a¤nity site with Kd of170^260 WM. It is important to note, that both ofthese values are greater for both resolved E1 and forits variants than those determined for the PDH com-plex by kinetic measurements in our lab [11] and byHennig et al. [37].

With the parental 1-lip and 3-lip PDH complexes,there is also a fast binding step, that howeveris followed by a slow isomerization, taking as longas 50^100 s before steady-state product releaseis achieved. This isomerization step may be phys-ical or perhaps even chemical, such as a ligand sub-stitution reaction, in which the Mg(II) achievesits best coordination geometry, that in turn alignsthe ThDP for highest activity. What is clear fromthe X-ray pictures of the ThDP enzymes is that theThDP ¢ts rather snugly into its fold. Any impair-ment of the optimal Mg(II) ligands could indeedchange the orientation of the coenzyme, and inturn the optimal juxtaposition of the catalyticgroups.

3.2.3. Regulation of the PDH complex and E1 bypyruvate

Both 1-lip and 3-lip PDH complexes could besaturated by pyruvate. The S0:5 = 0.21 mM (nH =1.1) and 0.22 mM (nH = 1.24), respectively [11]. TheEadie-Scatchard plots revealed weak positive cooper-ativity for both complexes (Fig. 7A presents data for1-lip PDHc) and for the C259S variant of 1-lipPDHc (data not presented), indicating that C259 isnot critically important for regulation by pyruvate.

However, the G231A substitution in the 1-lip PDHcdramatically altered the homotropic regulation bypyruvate, exhibiting weak negative cooperativity in-stead of the weak positive cooperativity seen with the1-lip PDHc (Fig. 7C).

In the presence of acetyl-CoA (0.39^0.65 mM), theproduct of the E2 reaction, the positive homotropiccooperativity for pyruvate increased, with nH of 1.4and 1.6 for the 1-lip and 3-lip PDH complexes, re-spectively (Fig. 7B presents data for 1-lip PDHc).Similar observations were made with the C259Nand C259S variants of 1-lip PDHc, suggesting thatC259 is not critical for the regulation of E1 activityby acetyl-CoA.

For the parental E1 and its cysteine variants(C120A, C575A, C610A, C654A and C770S), thepresence of 0.65 mM acetyl-CoA does not sig-ni¢cantly in£uence S0:5 for pyruvate, however theacetyl-CoA does increase the Hill coe¤cient to1.14^1.32 from 0.96^1.05 in its absence (see Fig. 8for the C575A variant). The kinetic data indicateweak positive cooperativity, suggesting that noneof the cysteine residues of E1 is essential for pyruvatebinding or for regulation of pyruvate bindingby acetyl-CoA [39]. The results also con¢rmedthe data from Bisswanger and Henning [41] forE. coli PDHc, indicating that the acetyl-CoA bind-ing site is located on the E1 component of the com-plex.

3.2.4. Current working hypothesis for regulation of E1by cofactors and pyruvate

The £uorescence binding data and the kinetic dataprovide excellent complementary evidence for thebinding mechanism of ThDP.Mg(II) as summarizedbelow:

�E1�2 � fThDP:Mg�II�g�K1�E1�2WfThDP:Mg�II�g�k2

k32

�E10�2WfThDP:Mg�II�g � ThDP�k3

k33

�E10�2WfThDP:Mg�II�g2

Substitution Fluorescence (resolved E1and variants)

Kinetic data from lag phase (1-lip PDHc, 3-lipPDHc and variants of 1-lip PDHc)

Information

WT Kd1 and Kd2 2 lag phases; yielding Kd1, k2, k32, k3, k33 two saturable sites;slow conformational changes

G231S Kd1 not Kd2 no lag phase one saturable site;no slow conformational changes

C259N Kd1 and Kd2 one lag phase at high ThDP two saturable sites;one slow conformational change



where Kd1 and Kd2 are the tighter and weaker disso-ciation constants for ThDP.Mg(II). According to theresults, the ¢rst ThDP.Mg(II) is bound tightly toall variants, providing the half-saturated E1 dimer.This is followed by a slow conformational equilibra-tion, only detected in the parental 1-lip and 3-lipPDH complexes and resolved E1. The secondThDP.Mg(II) can saturate the parental 1-lip PDHcand C259N variant, but not the G231S variant. Sincethe G231S variant cannot be saturated, it is perhapsnot surprising that the slow conformational change iseither non-existent, or cannot be detected. It is usefulto point out though that the magnitude of the rateconstant k3 reported for the parental 1-lip PDH com-plex (the second-order rate constant is many ordersof magnitude below that expected for a di¡usioncontrolled reaction) suggests that k3, k33 combine abinding event and a subsequent slow conformationalchange.

Since G231 is the second G in the GDG tripletconserved in all ThDP folds, and since the aspartatein the middle is a key ligand to the Mg(II), a veryplausible explanation is that ligand substitution reac-tions, replacing water around the Mg(II) by the pro-tein ligands is the ¢rst step in this reaction, followedrapidly by addition of ThDP. Any compromise in thecarefully crafted ligand environment would compro-mise ideal binding and activation by ThDP.Mg(II).These variants would be good candidates for X-raystudies once the structure of E1 is successfully solved,since they are likely to provide structural insight tothe activation process.

4. Conclusions

While considerable progress has been made duringthe past few years in our understanding of these reg-ulatory mechanisms, much remains to be done toprovide structural explanation for the kinetic andspectroscopic observations.

The di¤cult job of de¢ning a role for several ofthe amino acids near the ThDP still poses a chal-lenge. It is already clear that substrate and coenzymeactivation are complex processes that involve severalamino acid side chains. It is also important toemphasize that PDC that is devoid of the coopera-tivity (by virtue of substitutions at amino acids which

we now believe participate in substrate activation) isstill a functioning enzyme, just as is PDC from Zy-momonas mobilis. Substrate activation is by nomeans required for PDC activity to obtain. Whatappears to be confounding the issues is that largegroups covalently attached to C221 may indeed dis-tort the active center as well, perhaps by transmissionof the information along the same pathway as thatfollowed by interaction with pyruvate. On the otherhand, chemical reactions at only the substrate acti-vation site need not lead to total inactivation.Further delineation of the regulatory pathways willbe very rewarding, especially if some of the key var-iants lend themselves to high resolution structuralstudies.

Acknowledgements

The group at Rutgers is grateful to Dr. StefanHohmann for a fruitful collaboration leading to theidenti¢cation of cysteine 221 as the site of the initialsubstrate activation event.Supported at Rutgers byNIH-GM-50380 (to FJ) and the Rutgers UniversityBusch Fund, at She¤eld by the Biotechnology andBiological Sciences Research Council (UK, toJ.R.G.), and in Pittsburgh by NIH-GM-48195 (toWF).

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Documents

Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) – evidence for tertiary and quaternary interactions