Báo cáo khoa học: Reaction mechanisms of thiamin diphosphate enzymes: defining states of ionization and tautomerization of the cofactor at individual steps docx

MINIREVIEW Reaction mechanisms of thiamin diphosphate enzymes: defining states of ionization and tautomerization of the cofactor at individual steps Natalia S. Nemeria, Sumit Chakraborty, Anand Balakrishnan and Frank Jordan Department of Chemistry, Rutgers, The State University of New Jersey, Newark, NJ, USA Introduction Mindful of the fact that there are several reviews on the enzymology of thiamin diphosphate (ThDP, the vitamin B1 coenzyme; for structures of small molecules mentioned in the present review, see Fig. 1) available in the literature [1–15], the present review aims to con- centrate on the tautomeric and ionization states of ThDP on enzymes, which is a fascinating and, in some respects, perhaps unique aspect of thiamin enzymology. Keywords 1¢,4¢-iminopyrimidine tautomeric form of thiamin; benzaldehyde lyase; benzoylformate decarboxylase; CD; enamine intermediate; pyruvate decarboxylase; pyruvate dehydrogenase; thiamin diphosphate Correspondence N. S. Nemeria, 73 Warren Street, Newark, NJ 07102, USA Fax: +1 973 353 1264 Tel: +1 973 353 5727 E-mail: nemeria@rutgers.edu F. Jordan, 73 Warren Street, Newark, NJ 07102, USA Fax: +1 973 353 1264 Tel: +1 973 353 5470 E-mail: frjordan@rutgers.edu (Received 23 October 2008, revised 4 February 2009, accepted 9 February 2009) doi:10.1111/j.1742-4658.2009.06964.x We summarize the currently available information regarding the state of ionization and tautomerization of the 4¢-aminopyrimidine ring of the thia- mine diphosphate on enzymes requiring this coenzyme. This coenzyme forms a series of covalent intermediates with its substrates as an electro- philic catalyst, and the coenzyme itself also carries out intramolecular pro- ton transfers, which is virtually unprecedented in coenzyme chemistry. An understanding of the state of ionization and tautomerization of the 4¢-aminopyrimidine ring in each of these intermediates provides important details about proton movements during catalysis. CD spectroscopy, both steady-state and time-resolved, has proved crucial for obtaining this infor- mation because no other experimental method has provided such atomic detail so far. Abbreviations 3-PKB, (E)-4-(pyridine-3-yl)-2-oxo-3-butenoic acid; AcP ) , acetylphosphinate; AP, the canonical 4¢-aminopyrimidine tautomer of ThDP or its C2-substituted derivatives; APH + , the N1-protonated 4-aminopyrimidinium form of ThDP or its C2-substituted derivatives; BAL, benzaldehyde lyase; BFDC, benzoylformate decarboxylase; E1ec, the first component of the Escherichia coli pyruvate dehydrogenase complex; E1h, the first component of the human pyruvate dehydrogenase complex; GCL, glyoxylate carboligase; HBThDP, C2a-hydroxybenzylThDP, the adduct of benzaldehyde and ThDP; HEThDP, C2a-hydroxyethylThDP, the adduct of acetaldehyde and ThDP; IP, 1¢,4¢-iminopyrimidine tautomer of ThDP or its C2-substituted derivatives; LThDP, C2a-lactylThDP, the adduct of pyruvic acid and ThDP; MAP, methyl acetylphosphonate; MBP, methyl benzoylphosphonate; PAA, (E)-3-(pyridine-3-yl) acrylaldehyde; PLThDP, C2a-phosphonolactylThDP, the adduct of MAP and ThDP; POX, pyruvate oxidase from Lactobacillus plantarum; ThDP, thiamin diphosphate; TK, transketolase; Yl, C2-carbanion ⁄ ylide ⁄ carbene form conjugate base of ThDP; YPDC, yeast pyruvate decarboxylase from Saccharomyces cerevisiae. 2432 FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS This issue has come to the fore relatively recently, but its understanding is made more urgent and more sig- nificant by some recent X-ray crystal structure determi- nations of ThDP enzymes. Briefly, the question is related to the conundrum that any plausible mecha- nism suggested for ThDP-dependent enzymes, be they 2-oxoacid decarboxylases or carboligases [examples of a non-oxidative decarboxylase yeast pyruvate decar- boxylase (YPDC; EC 4.1.1.1), an oxidative decarboxyl- ase, the pyruvate dehydrogenase complex (EC 1.2.4.1), and a carboligase benzaldehyde lyase (BAL; EC 4.1.2.38) are given in Schemes 1–3], requires some proton transfer steps. On the basis of the accumulated understanding of enzyme mechanisms, such proton transfers are likely to be mediated by general acid ⁄ base catalysts, such as His, Asp and Glu, and perhaps Cys, Lys and Tyr, with the understanding that the enzyme active center could modulate the aqueous pK a of these side chains, as needed. Several groups, including our own [16], have spent considerable time trying to assign acid ⁄ base functions to such residues on ThDP enzymes, with limited suc- cess. Very recently, Yep et al. [17] carried out satura- tion mutagenesis experiments probing the function of two active center histidine residues (His70 and His281) on benzoylformate decarboxylase (BFDC; EC 4.1.1.7), long believed to participate in acid ⁄ base reactions [18]. Surprisingly, their results indicated that hydrophobic residues could replace the His281 with little penalty, and the His70Thr or His70Leu substitutions Scheme 1. Mechanism of yeast pyruvate decarboxylase YPDC. N S Me R2 Me HO CO 2 – N S Me R2 N S Me Me HO Me N S Me R2 N S Me R2 + yli de, Yl LThDP, IP Me O – enamine/ C2α-carbanion, AP(or APH + ) + + R1 R1 + – k 2 k 3 k 5 R1 = 4'-amino-2-methyl-5-pyrimidyl R2 = β-hydroxyethyldiphosphate OH S8-acetyldihydrolipoyl-E2 R2 C H 3 C O C O 2 - CO 2 k –MM R1 R1 R1 k 4 lipoyl-E2 2-AcThDP, AP (or APH + ) S S E2 SH S E2 CoASH CH 3 COSCoA dihydrolipoyl-E2 SHHS E2 E3 +FAD+NAD + N S Me R2 + R1 – k M M pyruvate. Michaelis complex k –2 HN N N S NH Me Me R2 H N N N S NH 2 Me Me H + 4'-aminopyrimidinium, APH + + 1',4'-iminopyrimidine, IP R2 N N N S NH 2 Me Me H 4'-aminopyrimidine, AP + R2 thiazolium -H1', pK 1' –H4' 1' 4' 2 3' + H –H4', pK 4' –H2, pK 2 Ke q K tautomerization H 3 COC MM, AP N S Me R2 Me HO H + R1 HEThDP, IP k 6 k –6 Scheme 2. Mechanism of E coli and human pyruvate dehydrogenase complex with role of ThDP. N. S. Nemeria et al. Enzyme-bound imino tautomer of thiamin diphosphate FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS 2433 only led to a 30-fold penalty on k cat ⁄ K m . A reason- able question in the interpretation of such findings is what is the appropriate contribution from His, Asp or Glu to reflect general acid ⁄ base reactivity on the enzyme? There appear to be two well-explored exam- ples that could provide benchmark values, although the precise interpretation of these numbers is not only risky, but also depends on the particular substitution used to arrive at them [19]: (a) serine proteases, where substitution of either His (a presumed general acid ⁄ base catalyst) or Ser (a nucleophilic catalyst) by Ala in the well-characterized Asp-His-Ser catalytic triad of subtilisin leads to an approximate 2 · 10 6 reduction in k cat , with little impact on k cat ⁄ K m [20] and (b) ketosteroid isomerase (EC 5.3.3.1), where sub- stitution of the catalytic Asp38 by Asn leads to a 10 5.6 decrease in k cat [21], whereas substitution of the same residue by Ala only reduced the k cat by 140 [22]. Complicating this issue on ThDP enzymes is that the pH dependence of the steady-state kinetic parame- ters does not provide clear evidence for the participa- tion of such residues in the rate-limiting step(s). For example, all potential active center acid ⁄ base residues were substituted on YPDC [16], with little perturbation of the pH dependence of such plots, perhaps with the exception of the substitution at the conserved gluta- mate. Therefore, the 100- to 500-fold reduction in steady-state kinetic constants could not be unequivo- cally attributed to acid ⁄ base function, whereas such numbers are certainly consistent with hydrogen-bond- ing interactions. Relevant to the issue of acid ⁄ base catalysis, the structure of two interesting ThDP-dependent lyases was solved with unusual characteristics. The enzyme BAL carries out reversible decomposition of (R)-ben- zoin to two molecules of benzaldehyde according to the mechanism given in Scheme 3; in the reverse direc- tion, the enzyme is a carboligase. The BAL structure reported contained only two acid ⁄ base residues sur- rounding the ThDP at the active center [23–25]: a highly conserved Glu50 within hydrogen-bonding dis- tance of the N1¢ atom of the 4¢-aminopyrimidine (AP) ring and a His29 residue. The residue His29 is too far from the thiazolium C2 atom to be of value in the first steps of the reaction and was suggested to have a function in removing the b-hydroxyl proton of the ThDP-bound benzoin to assist in releasing the first benzaldehyde molecule. In the authors’ view, this enzyme provides the clearest interpretation of the pH dependence of the steady-state kinetic parameters of any ThDP enzymes to date. There is a pK a = 5.3 at the acidic side of either the k cat -pH or k cat ⁄ K m -pH pro- file, almost certainly corresponding to the highly con- served glutamate residue [26]. With this information in hand, the pH dependence of kinetic parameters on YPDC could be re-examined, suggesting that the conserved glutamate affected the behavior similarly. The second case reported even greater surprises: the enzyme glyoxylate carboligase (GCL; EC 4.1.1.47) carries out a carboligation reaction after decarboxyl- ation of the first molecule of glyoxal to the enamine intermediate. This enzyme is not only devoid of acid ⁄ base groups at its active center within hydrogen-bond- ing distance of ThDP, but it is also lacking the highly conserved Glu and, in its place, there is a hydrophobic valine residue [27]. These two case studies suggest that our understand- ing of ThDP enzymes is not nearly as complete as was previously assumed, and certainly suggest that the N + S R 2 HO Ph N S R 2 N S R 2 Ph HO Ph HO N S R 2 ylide Mechanism of benzaldehyde lyase – C2α-carbanion/enamiine + R 1 R 1 + – k 2 HN N N S NH R 2 H N N N S NH 2 H + 4'-aminopyrimidinium + 1',4'-iminopyrimidine R 2 N N N S NH 2 4'-aminopyrimidine + R 2 thiazolium –H1' –H4' 1' 4' 2 3' k –2 R 1 R 1 N + S R 2 Ph OH R 1 + H k 1 /k –1 PhCHO HBThDP k –4 k 4 k –5 k 5 PhCHO AP APH + IP λ max 380 nm Ph Ph O OH Ph OH DDEThDP PhCHO PhCHO k 3 k –3 Ph = C 6 H 5 Scheme 3. Mechanism of benzaldehyde lyase. Enzyme-bound imino tautomer of thiamin diphosphate N. S. Nemeria et al. 2434 FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS ThDP cofactor has a much more dramatic impact on the reaction pathway than hitherto accepted. With results such as those described above, the coenzyme and its chemical reactivity need to be scrutinized from a newer vantage point. Early evidence indicating a catalytic function for the AP ring The chemistry and enzymology of ThDP is intimately dependent on three chemical moieties comprising the coenzyme: a thiazolium ring, a 4-aminopyrimidine ring and the diphosphate side chain (Fig. 1). From the large number of high-resolution X-ray structures available over the past 16 years, starting with the structures of transketolase [28] (TK; EC 2.2.1.1), pyru- vate oxidase [29] (POX; EC 1.2.3.3) from Lactobacil- lus plantarum and YPDC [30,31], it has become clear that the diphosphate serves to bind the cofactor to the protein. This is achieved via electrostatic bonds of the a and b phosphoryl group negative charges with the required Mg 2+ or Ca 2+ , the divalent metal serving as an anchor in a highly tailored environment with a universally conserved GDG recognition site and the diphosphate-Mg 2+ binding motif consisting of a GDG-X 26 -NN sequence of amino acids, as sug- gested by the Hawkins et al. [32]. As shown in a series of seminal studies by Breslow, the thiazolium ring is central to catalysis [33], as a result of its ability to form a key nucleophilic center at the C2 atom, the C2-carbanion ⁄ ylide or carbene, depending on one’s viewpoint with respect to the relative importance of the resonance contributions. The demonstration that the thiazolium C2H can undergo exchange with D 2 O, and that thiazolium salts per se, even in the absence of the AP ring, can induce benzoin condensations in a manner analogous to the cyanide ion catalyzed ben- zoin condensation, led to the proposal of the pathway involving thiazolium-bound covalent intermediates, as also shown in Schemes 1–3. Thus, is there anything else to thiamin catalysis? It was reported that the pro- tein environment of YPDC provides a catalytic rate acceleration of 10 12 –10 13 [34]. Is this simply a result of juxtaposition of amino acid side chains to provide the general acid ⁄ base catalysis, or an enzymatic sol- vent effect [10,14] and does it include a contribution Fig. 1. Compounds under discussion. N. S. Nemeria et al. Enzyme-bound imino tautomer of thiamin diphosphate FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS 2435 from the special properties of ThDP when enzyme bound? Starting in the 1960s, Schellenberger and his princi- pal associate Hu ¨ bner, and their colleagues in Halle, examined the role of the AP ring [8]. Most notably, they undertook de novo synthesis of thiamin diphos- phate analogs replacing each of the three nitrogen atoms of the AP ring in turn. They then tested each of these deaza analogs for coenzyme activity on a number of enzymes. The results clearly indicated that the N1¢ atom and the N4 ¢ -amino group are absolutely required, with the N3¢ atom to a lesser extent. On the basis of application of this powerful probe to a num- ber of ThDP enzymes, the group from Halle made the totally reasonable suggestion that the AP ring has cat- alytic role, and does not serve simply as an anchor to hold the coenzyme in place. The idea was further elab- orated at Rutgers with a synthetic model in which the mobile proton at the N1 ¢ position (the principal site of first protonation of the AP) was replaced by a methyl group, creating N1¢-methylthiaminium and N1¢-meth- ylpyrimidinium salts, consequently demonstrating that the positive charge installed at the N1¢ position con- verted the amino group to a weak acid with a pK a of almost 12–12.5 in aqueous solution [35]. This raised the possibility of the existence of the 1¢,4¢-iminopyrimi- dine (IP) tautomer for the first time. This was impor- tant because the earlier model for AP reactivity typically assumed that the amino group, as a base, would accept a proton. As more information became available about protonation sites in aminopyridines and aminopyrimidines, such as the nucleic bases, it became clear that ring nitrogen protonation is pre- ferred over protonation of the exocyclic amino group. The hypothesis suggesting the AP moiety as an impor- tant contributor to catalysis and the possibility for its participation in acid ⁄ base catalysis [35] has gained wider acceptance subsequent to the appearance of the X-ray structures of ThDP enzymes. The following gen- eralizations could be made on the basis of structural observations that hold in virtually all of the ThDP enzyme structures: (a) strong hydrogen bonds from the protein to both the N1¢ atom (via a conserved Glu with the exception of the enzyme GCL so far) and to the N4¢-amino nitrogen atom on the side of the N3¢ atom of the ring; (b) an unusual V conformation (describing the disposition of the AP and thiazolium rings with respect to the bridging methylene group) [36] rarely observed in model ThDP structures [37], and predicted to be in a high energy region in van der Waals conformational maps [38]; and (c) a surprisingly short < 3.5 A ˚ distance between the AP amino nitro- gen atom and the thiazolium C2 atom. Detection of intermediates on ThDP enzymes in solution A number of methods now exists to monitor the kinetic fate of each covalent ThDP-substrate interme- diate along the catalytic cycle of various ThDP enzymes represented by examples in Schemes 1–3 [10,14,15,39]. The three ThDP-bound intermediates in Scheme 1 could be classified as: a pre-decarboxylation intermediate C2a-lactylThDP (LThDP) or its analogs, the first post-decarboxylation intermediate (the enam- ine), and the second post-decarboxylation intermediate C2a-hydroxyethylThDP (HEThDP) or its analogs. The last one could also be construed as a product-ThDP adduct for decarboxylases. A distinguishing feature of these three intermediates is that the first (LThDP) and third (HEThDP) have tetrahedral substitution at the C2a atom, whereas the enamine being conjugated should be trigonal planar at this position. Below, a brief summary is given of the presence of various ThDP intermediates on the enzymes, and the informa- tion that has emerged regarding the state of ionization and tautomerization of the AP ring on these intermedi- ates. Understanding these issues is important with respect to monitoring proton movements during catalysis. A convenient way to view ThDP-related and ThDP- bound intermediates is to classify them as pre-, or post-substrate (or substrate analog) binding. ThDP-related intermediates prior to substrate addition For reasons mentioned earlier, during the recent past, a need arose for the direct detection of various inter- mediates shown in Schemes 1–3. Although the NMR method developed by Tittmann and Hu ¨ bner [39] could identify most of the covalent ThDP-bound substrates and products on the pathway, the tautomeric forms and ionization states of the 4¢-aminopyrimidine ring along the reaction pathway and under the reaction conditions remained to be elucidated. The AP form of ThDP The signature for this species is a negative CD band centered near 320–330 nm and is well illustrated by the enzyme BAL (Fig. 2). Although this CD band has long been observed on the enzyme TK [40], it had been suggested to be the result of a charge transfer transi- tion between ThDP and an amino acid side chain on TK, although early reports attributed it to the ThDP itself. A number of studies at Rutgers on YPDC and Enzyme-bound imino tautomer of thiamin diphosphate N. S. Nemeria et al. 2436 FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS the first component of the Escherichia coli pyruvate dehydrogenase complex (E1ec; EC 1.2.4.1.) and their variants, as well as chemical model studies, strongly suggest that this CD band is due to a charge transfer transition between the AP ring as donor and the thia- zolium ring as acceptor [41,42]. This CD band has now been observed on a number of ThDP enzymes (Table 1) and its detection strongly depends on pH and, to a significant extent, on the enzyme environ- ment. The IP form of ThDP [41–45] The notion that the AP could exist in the IP tauto- meric form was suggested earlier by models attempting to mimic the reactivity of such a tautomer. In the N1¢- methylpyrimidinium, the pK a of the exocyclic amine is reduced to approximately 12–12.5 [35,45], offering rationalization for the presence of conserved glutamate as a catalyst for the amino–imino tautomerization. The positive charge on the 4¢-aminopyrimidinium ring also induced differential exchange rates for the two amino protons and the exchange was found to be buf- fer catalyzed [46]. The first evidence for the possibility that the IP tautomer may have a spectroscopic signa- ture was found on the slow E477Q variant of YPDC [43]. Inspired by these results, the old models were dusted off and, in a series of chemical model studies, Jordan et al. [43] and later Baykal et al. [45] showed that an appropriate chemical model for the IP will give rise to a UV absorption in the 300–310 nm range. Ser- endipitously, the 15 N chemical shifts of the three species on the left hand side of Schemes 1 and 2, the two neutral and one positively charged forms of the Fig. 2. CD detection of the AP form of ThDP on BAL. Inset: pH dependence of the amplitude of the band for the AP form of ThDP. Determination of pK a for the ([AP]+[IP]) ⁄ [APH + ] equilibrium on BAL [45]. Table 1. Assignment of the state of tautomerization of ThDP during the reaction pathway. ND, not detected. ThDP intermediates IP positive CD, 300–314 nm AP negative CD, 320–330 nm References ThDP E1h E1h [26,27,44] POX POX V51D GCL BAL (pH > 6.0) BFDC (pH > 7.0) Michaelis–Menten complex ND E91D YPDC-MAP [42,44,52] E51D YPDC-MAP YPDC-AcP - E571A E1ec-Py E401K E1ec-Py POX-AcP ) Pre-decarboxylation reaction intermediate analog E91D YPDC-MAP [14,25,42,44,58] E51D YPDC-MAP YPDC-AcP ) E1ec-MAP E1ec-AcP ) E1h-AcP ) POX-AcP ) BAL-BF BAL-PPy BFDC-MBP BAL-MBP Enamine (stopped-flow photodiode array) ND ND Post-decarboxylation BAL-PAA ND [61] YPDC+acetaldehyde N. S. Nemeria et al. Enzyme-bound imino tautomer of thiamin diphosphate FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS 2437 AP, are quite distinct [45]; early 15 N NMR experi- ments on this issue were conducted by Cain et al. [47]. The recognition that the CD bands corresponding to the AP and IP forms have different phases enables the simultaneous observation of the two tautomeric forms, notwithstanding the proximity of the bands to each other, and also make CD the method of choice for such studies. The signature for this IP species is a posi- tive CD band centered near 300–314 nm (Table 1) and is well illustrated on the first component of the human pyruvate dehydrogenase complex (E1h), where both the IP and AP tautomeric forms can be observed simultaneously (Fig. 3). To the authors’ knowledge, no electronic absorption characteristic of the N1-protonated 4-aminopyrimidinium form of ThDP or its C2-substituted derivatives (APH + ) or the ylide (Yl) has yet been proposed. Determination of pK a for the enzyme-bound APH + form [26] As the pH is lowered, the amplitude of the band for the AP form diminishes and titrates with an apparent pK a = 7.42 for the ([AP]+[IP]) ⁄ [APH + ] equilibrium on BAL (Fig. 2). This pK a in water for ThDP is 4.85 [47], whereas, on the enzymes, it is in the range 5.6–7.5 (Table 2) [26]. From the data provided in Table 2, it was concluded that the pK a for the APH + coincides with the pH of optimum activity for each enzyme, indicating that all three forms (IP, AP and APH + ) must be readily accessible during the catalytic cycle. The pK a elevation on the enzymes could be rational- ized by the presence of the highly conserved glutamate near the N1¢ position of ThDP, which would tend to make the AP ring more basic. The tautomeric equilib- rium constant K tautomer , in conjunction with the pK a led to a novel insight regarding ThDP catalysis, best viewed by the thermodynamic box for enzymes that are not substrate activated (Scheme 2, left hand side), such as E1h and POX from L. plantarum. For these enzymes, both the IP and AP forms could be moni- tored over a wide pH range, providing both pK a and K tautomer within reasonable error limits. The equilibria shown in Schemes 1 and 2 are valid prior to addition of substrate and lead to the tantalizing conclusions: (a) on POX and E1h, pK 1¢ and pK 4¢ have similar mag- nitudes; the enzymes shifted the pK 4 ¢ from 12 in water [35] to 5.6 and 7.0, respectively (see left triangle in Schemes 1 and 2), and (b) with a known forward rate constant from APH + to the Yl of approximately 50 s )1 determined for E1h [48], and assuming a diffu- sion-controlled reverse protonation rate constant of 10 10 s )1 Æm )1 (giving a pK 2 of 8.3 on E1h compared to an estimate in water of 17–19) [49], it is possible to speculate about the right triangle in Schemes 1 and 2. The most important conclusion is that the proton- transfer equilibrium constant for [IP] ⁄ [Yl] is 10 1 –10 2 on E1h. These thermodynamic parameters are the first estimates on any ThDP enzyme and should be gener- ally applicable to ThDP enzymes. The results also suggest conditions under which a significant fraction of the thiazolium ring may be in the conjugate base ylide form. The results provided in Table 2 also indicate that, when the AP form is observable, below the pK a , the APH + form likely exists, which comprises a form with no known spectroscopic signature as far as we aware. The C2-carbanion ⁄ ylide ⁄ carbene According to the findings of Breslow, proton loss at the thiazolium C2 position is required to initiate the catalytic cycle. In 1997, there were two studies reporting significant implications regarding this issue: (a) Arduengo et al. [50] showed that the conjugate Fig. 3. CD spectra of E1h titrated with ThDP. The spectra revealed the presence of both the IP (at 305 nm) and AP (at 330 nm) tauto- meric forms of ThDP [44]. Table 2. Correlation of pKa of enzyme bound APH+ and pH opti- mum of enzyme activity. Enzyme pH optimal activity pKa for ([AP] + [IP]) ⁄ (APH + ) BAL 6.5–7.5 7.42 ± 0.02 BFDC 6.0–8.5 7.54 ± 0.11 POX 5.6–6.2 5.56 ± 0.03 E1h 7.0–7.5 7.07 ± 0.07 Enzyme-bound imino tautomer of thiamin diphosphate N. S. Nemeria et al. 2438 FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS bases of imidazolium and indeed of thiazolium salts could be generated and it was possible to study their structure by NMR methods. In the intervening years, some of these carbenes have been used in organometal- lic reactions, including olefin metathesis. Arduengo et al. [50] showed that the 13 C chemical shift of the C2 resonance shifted from 157 to 253 p.p.m. on conver- sion of their model thiazolium compound to its conju- gate base, thereby providing the all important guide for future attempts to observe the ylide. (b) At the same time, the group at Halle reported 13 C measure- ments with specifically-labeled ThDP, according to which, on the YPDC, the thiazolium ring C2H of bound ThDP is in its undissociated state, both in the absence and presence of the substrate activator surro- gate pyruvamide (this enzyme has long been known to be substrate activated); in other words, no evidence was found for the presence of the conjugate base in the activated or unactivated forms of YPDC [51]. It is important to emphasize that determination of the state of ionization and tautomerization of enzyme- bound ThDP by solution NMR methods poses several challenges, both in the absence and presence of substit- uents at the C2 atom: (a) the size of ThDP enzymes (> 120 kDa) leads to broadened lines; (b) for many ThDP enzymes, it is difficult to reversibly remove ThDP and replace it with labeled coenzyme; and (c) de novo synthesis required for specific labeling of ThDP is time consuming and expensive. Thiamin-bound intermediates with substrate or substrate analog present The Michaelis–Menten complex Our earliest detection of an Michaelis–Menten com- plex was on addition of a substrate analog methyl acetylphosphonate (MAP) and acetylphosphinate (AcP ) ) to several ThDP enzymes (Table 1). An exam- ple is shown with AcP ) added to YPDC (Fig. 4) leading to a negative CD band at approximately 325– 335 nm, which is very reminiscent of the band observed for the AP form [44]. Similar results were also seen when low concentra- tions of pyruvate were added to E1ec [42]. Clear evi- dence for the formation of the Michaelis–Menten complex with a negative CD band near 320 nm was also provided when adding pyruvate to the ‘inner loop’ E1ec variants [52]. Especially valuable support for the claim that the Michaelis–Menten complex was indeed being detected is provided by kinetic measurements: stopped-flow photodiode array spectra in the absorp- tion mode, as well as stopped-flow CD spectra at the appropriate wavelength, showed formation of the absorbance ⁄ CD band attributed to Michaelis–Menten complex formation, within the dead-time of the stopped-flow instruments (< 1 ms), as expected of a noncovalent Michaelis–Menten complex [52]. From these results, we conclude that the Michaelis– Menten complex is in the AP form. The covalent substrate-ThDP pre-decarboxylation complex (LThDP and analogs) Observation of pre-decarboxylation intermediate derived from aromatic substrates In some favorable cases, such as with BAL, the posi- tive CD band at 300–314 nm (Table 1) for the pre- decarboxylation intermediate (via the IP form) could be observed from the slow substrates benzoylformate or phenylpyruvic acid [53]. This is plausible because BAL, although a carboligase ⁄ lyase enzyme, also cata- lyzes the decarboxylation of aromatic 2-oxoacids, albeit very slowly. Observation of stable pre-decarboxylation intermediates derived from substrate analog phosphonates and phosphinates The initial identification of the IP form (positive CD band, 300–314 nm) resulted from formation of a stable pre-decarboxylation adduct of ThDP with: (a) MAP [41,42] or AcP ) [44], with pyruvate-specific enzymes and (b) the aromatic 2-oxo acid analog methyl ben- zoylphosphonate (MBP) with BFDC and BAL [25,53], Fig. 4. CD spectra of YPDC in the presence of AcP ) . The spectra revealed the presence of the Michaelis–Menten complex in the AP form (325–335 nm) and of the 1¢,4¢-iminophosphinolactyl-ThDP covalent pre-decarboxylation intermediate in IP form (302 nm). Inset: dependence of 1¢,4¢-iminophosphinolactyl-ThDP formation at 302 nm on [AcP ) ] [44]. N. S. Nemeria et al. Enzyme-bound imino tautomer of thiamin diphosphate FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS 2439 according to Scheme 4. With six ThDP enzymes tested so far (Table 1), the IP form appeared on the stopped- flow time scale (either absorption or CD mode; for the E1ec reaction with AcP ) ; see Fig. 5, top). The reaction is efficiently catalyzed by all of the enzymes tested (for E1h with AcP ) , see Fig. 5, bottom; Scheme 4). An important additional finding is shown in Fig. 4, result- ing from mixing YPDC and AcP ) [44]: because we are observing evidence for the coexistence of the Michael- is–Menten complex and the covalent pre-decarboxyl- ation intermediate, the results are consistent with ‘alternating active site reactivity’ in a functional dimer, as suggested for YPDC and BFDC [54–56]. We sug- gested that, although one active center catalyzes the pre-decarboxylation step, the other catalyzes the post- decarboxylation events [55,56,57]. Formation of C2a-phosphonomandelylThDP on BFDC from MBP and ThDP was also confirmed in solution (FT-MS) [58], and that of C2a-phospho- nolactylThDP (from MAP.ThDP) by X-ray methods on E1ec [59] and POX [60]. Observation of pre-decarboxylation adducts of ThDP with chromophoric substrate analogs Recently, in three enzymes, YPDC, BFDC [58,61] and BAL [53], the formation of the pre-decarboxylation adduct formed with ThDP from a chromophoric sub- strate analog (E)-2-oxo-4(pyridine-3-yl)-3-butenoic acid (3-PKB) (as well as its ortho- and para isomers) was also observed. In a series of studies on BAL [53], BFDC [61] and YPDC (Fig. 6), the compound 3-PKB provided outstanding information about the rates of formation of two important intermediates, the pre- decarboxylation LThDP analog and the enamine, which were not readily available from other experiments. At the same time, using (E)-3(pyridine-3- yl)-2-propenal (PAA, the product of decarboxylation of 3-PKB), provided not only information about the second post-decarboxylation intermediate, but also enabled us to assign the IP tautomeric form to both tetrahedral, LThDP and HEThDP analogs (see below). The first post-decarboxylation intermediate: the enamine ⁄ C2a-carbanion According to Schemes 1 and 2, the enamine is the only covalent thiamin-bound intermediate capable of being conjugated. Electronic spectral observation of the enzyme-bound enamine derived from aliphatic substrates is difficult due to the expected k max near 290–295 nm, according to thiazolium-based models [10,14]. With YPDC, BFDC and BAL, the enamine could be observed directly near 430 nm with 3-PKB as alter- nate substrate, as shown in Fig. 6 for YPDC. The enamine intermediate derived from benzoylfor- mate has been observed directly on the enzyme BFDC at 390 nm [61]. We had modeled this enamine with a k max of 380 nm) [10,14]. When BFDC was reacted with the benzaldehyde product, there was absorbance (and a CD band) at 390 nm, as predicted by the chemical models, but no CD band was evident in the 300–310 nm region, suggesting that the enam- ine is not in the IP form [61]. Also, when (R)-benzoin was added to BAL, the same CD band was formed at 390 nm, indicating the slow release of the first benzaldehyde, and the stability of the enamine in the forward direction [53]. These experiments provided fundamental information: (a) the ‘real’ enamine could Scheme 4. Mechanism of formation of LThDP and analogue adducts. Enzyme-bound imino tautomer of thiamin diphosphate N. S. Nemeria et al. 2440 FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS be observed (due to its long k max at 390 nm) for the first time derived from benzoin or benzaldehyde; (b) the enamine may be in its APH + form, but not in its IP form; and (c) because it gives rise to a CD signal, the enamine is chiral on the enzyme by virtue of the chirality induced by the enzyme, even though it is planar and conjugated. The enamine has also been detected indirectly using the Tittmann and Hu ¨ bner method [39]. The method is demonstrated with the E401K active center inner loop variant of the E1ec (Fig. 7), where we used the synthetic [C2,C6¢- 13 C 2 ]ThDP enabling measurement of the rate of enamine formation via HEThDP (unpublished results). The labeled ThDP allowed observation of only those protons directly attached to 13 C nuclei, simplifying analysis in this otherwise busy aromatic region, espe- cially for the pyruvate dehydrogenase complex, in which there are three additional aromatic moieties (FAD, NADH, CoA). Accumulation of the enamine ⁄ HEThDP, but not of LThDP, suggests that decarboxylation is faster than LThDP formation. Furthermore, for the E401K E1ec variant, assembly to the complex appears to accelerate the rate by a modest factor (Fig. 7). The second post-decarboxylation intermediate, the product-ThDP complex (HEThDP, C2a-hydroxy- benzylThDP) Clear evidence was obtained for HEThDP analog for- mation from reacting PAA (i.e. the product of decar- boxylation of 3-PKB) with BAL or BFDC [61]. The structure of BFDC with both PAA and 3-PKB was solved to high resolution [61]. The structure with PAA clearly indicated: (a) covalent binding to ThDP as the C2a-hydroxymethyl derivative with the vinylpyridyl substituent attached to the C2a atom, (b) a tetrahedral rather than trigonal environment at that atom because 10 4 3 2 Ellipticity (mdeg) 1 0 0123 Time (s) 45 8 6 4 2 Ellipticity (mdeg) Ellipticity (mdeg) 0 –2 –4 –6 300 320 340 330 nm 305 nm 1–150 µ M AcP [AcP]/[E1 h] (µ M/µM) 8 6 4 2 0 012345678910 360 Wavelen g th (nm) 380 400 420 440 Fig. 5. Formation of the pre-decarboxylation intermediate on the PDHc-E1 component from AcP ) . CD detection of the covalent 1¢,4¢-iminophosphinolactyl-ThDP intermediate on E1h from acetyl- phosphinate (bottom) and the rate of 1¢,4¢- iminophosphinolactyl- ThDP formation on E1ec by stopped-flow CD (top). Rate constants of k 1 = 4.44 ± 0.34 s )1 and k 2 = 0.593 ± 0.064 s )1 were calculated [44]. Wavelen g th (nm) 400 450 500 550 600 Relative absorbance 0.00 0.05 0.10 0.15 0.20 0.25 LThDP analogue ( ma x 473 nm ) Enamine ( ma x 435 nm ) Time (s) 0 10 20 30 40 50 60 7 0 Concentration ( M ) 0 2 4 6 8 10 12 14 16 18 [ES] Enamine LThDP analogue k 2 = 0.507 + 0.002 s –1 k 3 = 0.118 + 0.013 s –1 Fig. 6. Reaction of YPDC with 3-PKB. Left: direct observation of the enamine at 435 nm on YPDC derived from 3-PKB by stopped-flow pho- todiode array spectroscopy. Right: time course of intermediate formation after deconvolution of the spectrum. (S. Chakraborty, unpublished data). N. S. Nemeria et al. Enzyme-bound imino tautomer of thiamin diphosphate FEBS Journal 276 (2009) 2432–2446 ª 2009 The Authors Journal compilation ª 2009 FEBS 2441 [...]... assignment than to the IP form of HEThDP (Scheme 2) 2-Acetylthiamin diphosphate and the C2a-hydroxyethylidene cation radical There is no evidence yet regarding their state of tautomerization ⁄ protonation 2442 Assignment of the state of ionization and tautomerization to each intermediate on the pathway Perhaps our most significant observation is that, even in the absence of pyruvate, both the AP and IP forms... complex The results suggest that, for several steps, there are proton transfers in the reaction pathway that are required to ensure the presence of the appropriate tautomeric form for the intermediate This is evident from Scheme 2, where the tautomeric ⁄ ionization state of the AP is assigned to each intermediate Prospects Further examples, and alternative methods to confirm the electronic spectroscopic... substituent and the 4¢-imino nitrogen under these conditions Table 1 summarizes the nature of the tautomeric form suggested for all of the intermediates on the ThDP pathways and provides the supporting evidence for them Curiously, the mechanisms could be expounded invoking solely the IP and APH+ forms, whereas, to date, the canonical AP form has only been identified in the Michaelis–Menten complex The results... in its IP form We further suggest that the 2-acylThDP and the C2a-hydroxyethylideneThDP radical also are not in the IP form on account of the hybridization at C2a in these intermediates An asymmetry of active centers is revealed by several findings: (a) on POX and E1h (Fig 3), the IP and AP forms of ThDP coexist even in the absence of substrates; (b) on POX and YPDC (Fig 4) in the presence AcP), one... there is no conserved glutamate present? Fourth, what is the energetic cost to the enzyme of stabilizing the IP form at the active center and from where is that energy obtained? Fifth, does the simultaneous presence of the IP and AP forms on POX and E1h imply half -of- the- sites reactivity? Finally, where both the IP and AP forms are present simultaneously, is it the reflection of the so-called ‘proton wire’... Sixty years of thiamin diphosphate biochemistry Biochim Biophys Acta 1385, 177–186 9 Jordan F (1999) Interplay of organic and biological chemistry in understanding coenzyme mechanisms: example of thiamin diphosphate- dependent decarboxylations of 2-oxo acids FEBS Lett 457, 298–301 10 Jordan F (2003) Current mechanistic understanding of thiamin diphosphate- dependent enzymatic reactions Nat Prod Rep 20,... pyruvate; 30 s after addition of pyruvate at 25 °C Bottom: time course of HEThDP formation by E401K E1ec, and by the same variant reconstituted with the E2ec and E3ec components To either E401K E1ec or PDHc reconstituted with this variant [C2,C6-13C2]ThDP and pyruvate were added and, at the indicated times, the reaction was quenched into 12.5% TCA in 1 M DCl (A Balakrishnan, unpublished data) the planar... the APH+ and the ylide forms As with any novel finding, the observation of the 1¢,4¢-imino tautomeric form on the addition of ThDP itself to the enzymes, even in the absence of substrate or substrate analog, also raises many interesting questions, some of which are summarized below First, why does the negative CD band at 320– 330 nm correspond to the AP form observed in some and but not in other enzymes?... on YPDC and BFDC (also a homotetrameric ThDP enzyme) We note that there are several recent examples in the literature, both from our own work and that of others, suggesting that, with tetrahedral substitution at the C2a atom, the C2-C2a bond may be out of the plane of the thiazolium ring [25,53,58,59] This certainly suggests, but does not prove, that there is van der Waals repulsion between the C2a... the pyridyl and thiazolium rings are not coplanar with each other, thereby ruling out the enamine [61] Fig 9 Stopped-flow photodiode array spectra of YPDC with product of pyruvate decarboxylation, acetaldehyde Spectra show formation of the IP form of HEThDP (maximum at 310 nm) (S Chakraborty, unpublished data) Addition of phosphinate or phosphonate analogs of pyruvate or benzoylformate to the seven enzymes . MINIREVIEW Reaction mechanisms of thiamin diphosphate enzymes: defining states of ionization and tautomerization of the cofactor at individual steps Natalia S. Nemeria, Sumit Chakraborty, Anand Balakrishnan. of the presence of various ThDP intermediates on the enzymes, and the informa- tion that has emerged regarding the state of ionization and tautomerization of the AP ring on these intermedi- ates for the presence of the conjugate base in the activated or unactivated forms of YPDC [51]. It is important to emphasize that determination of the state of ionization and tautomerization of enzyme- bound