REVIEW ARTICLE Catalyzing separation of carbon dioxide in thiamin diphosphate-promoted decarboxylation Ronald Kluger and Steven Rathgeber Davenport Chemical Laboratories, Department of Chemistry, University of Toronto, Canada Decarboxylases and intermediates Thiamin diphosphate (ThDP) is a cofactor that promotes the decarboxylation of 2-ketoacids through formation of covalent derivatives between its C2 thia- zolium and the carbonyl of the substrate. Combina- tion of a protein and ThDP in a holoenzyme provides substrate specificity and the general enzymic advantage of reduced translational entropy that favors addition processes [1,2]. The covalent interme- diate undergoes cleavage of a bond to a carboxylate group derived from the 2-ketoacid, resulting in production of carbon dioxide. This also produces a residual acyl anion equivalent [3–6] with a delocalized structure that can also be represented as a neutral enamine. The sequence is illustrated for the decarbox- ylation of pyruvic acid by pyruvate decarboxylases in Scheme 1. Protonation at the basic carbon and elimination of ThDP leads to formation of an aldehyde (giving a net substitution of a proton for carbon dioxide). Oxidation of the same intermediate would yield an acid, while reaction with a carbonyl carbon gives a condensation product. The general route is based on concepts origi- nally developed by Breslow [7–9] based on studies of model compounds related to ThDP. Details of reaction patterns within that pathway reveal previously unrec- ognized aspects of enzymic catalysis [3,10]. Synthetic analogs of the covalent intermediates have been prepared and studied in order to arrive at a quantitative understanding of the separate functions of the cofactor and protein [11,12]. Spectroscopic analysis of the conjugates of thiamin and ketoacids has enabled specific and quantitative identification of the coenzyme derivatives bound to proteins in enzymic reactions [13–15]. Keywords active site; benzoylformate decarboxylase; carbanion; catalysis; decarboxylation; diffusion; fragmentation; pre-association; thiamin; thiamin diphosphate Correspondence R. Kluger, Davenport Chemical Laboratories, Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada Fax: +1 416 978 8775 Tel: +1 416 978 3582 E-mail: rkluger@chem.utoronto.ca (Received 22 July 2008, revised 2 October 2008, accepted 10 October 2008) doi:10.1111/j.1742-4658.2008.06739.x Thiamin diphosphate-dependent decarboxylases form addition intermedi- ates between thiamin diphosphate (ThDP) and 2-ketoacids. Although it appears that the intermediate should react without the intervention of cata- lysts, evidence has clearly shown that Brønsted acid catalysis occurs through a pre-associated system. This can promote separation of carbon dioxide from the residual carbanion by protonation of the carbanion. Proteins operate through pre-association and may readily promote the separation of carbon dioxide by protonating or oxidizing the nascent carb- anion. Alternatively, a nucleophilic side chain may trap carbon dioxide as an unstable hemi-carbonate. Mutagenesis experiments by others have shown that enhanced activity due to the protein in the presence of thiamin diphosphate does not depend on the presence of any one proton donor, consistent with pooled activity within the active site. This form of catalysis has not been widely recognized, but should be considered an integral aspect of enzyme-promoted decarboxylation. Abbreviations AHAS, acetohydroxy acid synthase; BFD, benzoylformate decarboxylase; HBnTh, 2-(1-hydroxybenzyl) thiamine; HBnThDP, 2-(1-hydroxybenzyl) thiamin diphosphate; MTh, a-mandelyl-thiamin; ThDP, thiamin diphosphate. FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS 6089 There are significant differences between the reac- tivity of synthetic intermediate analogs and the corre- sponding intermediates in enzymic systems, and these can reveal the specific role of the protein [4,5,12,16]. Quantitative differences in the decarboxylation of the conjugates of thiamin and 2-ketoacids provide impor- tant insights into the role of the protein as a catalyst in the decarboxylation step of an enzyme for which reactions are considerably faster than the comparable unimolecular reactivity of the synthetic intermediates [12,17,18]. Based on rate measurements in catalytic systems, we have recently proposed that the proteins increase the rates of decarboxylation of ThDP-derived intermediates of 2-ketoacids through their inherent ability to facilitate diffusion of carbon dioxide away from the ThDP-derived intermediate, avoiding the significant reverse reaction that is normally an inherent part of the non-enzymic reaction [3,18,19]. The rate constant for decarboxylation of a-lactyl- thiamin, the simplied analog of a-lactyl-thiamin diphosphate in Scheme 1, is approximately 10 6 times smaller than the typical k cat value for pyruvate decar- boxylase [12]. However, there is no site in the likely transition state for decarboxylation associated with the formation of carbon dioxide that would be stabilized by specific interaction with the protein. Therefore, we would not expect any groups on the protein to affect the rate, but the rate acceleration is clearly significant. Similarly, the intermediate analog for benzoylformate decarboxylase (BFD), the conjugate of thiamin and benzoylformate, a-mandelyl-thiamin (MTh) (Fig. 1), undergoes decarboxylation in neutral solution with a rate constant that is also approximately 10 6 times smaller than the k cat for BFD [11]. Catalysis by desolvation Crosby and Lienhard produced a simplified model for the conjugate of pyruvate and ThDP, 2-(1-carboxy- hydroxyethyl)-3,4-dimethylthiazolium chloride) [20]. They noted that its decarboxylation rate constant is at least 10 5 times smaller than that of the likely enzyme- bound intermediate derived from a-lactyl-thiamin diphosphate. They suggest that, instead of acid ⁄ base catalysis, which would not facilitate the decarboxyl- ation step, the enzyme could transfer the intermediate into an environment with reduced polarity. They sup- port this with evidence that the model intermediate’s rate of decarboxylation is much greater in solvents with reduced polarity. Despite the clear change in reactivity in low-polarity solvents, this hypothesis presents some difficulties. BFD has a similar intermediate and also shows rate acceleration of the conjugate of its substrate and ThDP, but has a very hydrophilic binding site for the substrate and coenzyme [3,21–23]. Thus, there is no obvious way to promote the decarboxylation step in general. Oka fragmentation of 2-(1-hydroxyben- zyl)-thiamin The product of decarboxylation of the conjugate of ThDP and benzoylformate is 2-(1-hydroxybenzyl) thia- min diphosphate (HBnThDP). Analysis of the prod- ucts in this reaction revealed that the C2a conjugate base of HBnTh undergoes a reaction that destroys thi- amin by a very rapid process that splits the pyrimidine and thiazolium portions (Scheme 2) [16,24]. Fig. 1. The structure of a-mandelyl-thiamin (MTh), an accurate reactivity model of the conjugate formed from ThDP and benzoyl- formate. Scheme 1. Covalent intermediates derived from thiamin diphosphate in the decarboxylation of pyruvate. Catalyzing separation of carbon dioxide R. Kluger and S. Rathgeber 6090 FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS The same products were originally observed by Oka et al. during an attempt to catalyze condensation of benzoin with thiamin promoted by a tertiary amine in ethanol [25]. In that study, it is likely that HBnTh fragmented after it formed. Our study of the products of the spontaneous reaction of MTh revealed that fragmentation is the specific result of a proton being removed from C2a, a process that is subject to general base catalysis; therefore, it must be the rate-determin- ing step or a component of that step. Removal of the proton from carbon rather than from the hydroxyl group (which would lead to generation of thiamin and benzaldehyde) is highly favored under conditions where the pyridimine is protonated or has a positive charge induced by alkylation [26,27]. The expected ionization of the C2a hydroxyl of HBnTh, followed by formation of benzaldehyde and thiamin, only occurs in more basic solutions and is subject only to specific base catalysis by the solvent lyate ion [26]. The occurrence of fragmentation of HBnTh is readily detected by observing the unique absorbance band at 328 nm that arises from the phe- nyl thiazole ketone product [25]. The unimolecular rate constant for fragmentation of HBnTh is approximately 10 4 s )1 at 30 °C, which is approximately 100 times larger than the k cat of BFD. Thus, the enzyme appears to accelerate decarboxylation of MThDP and to slow fragmentation of the anion derived from HBnThDP. The C2a conjugate base of HBnTh from decarboxylation – fragmentation and its implications Decarboxylation of MTh will produce the conjugate base at C2a of HBnTh as the immediate product, along with carbon dioxide. In the presence of low con- centrations of acid components of phosphate or ace- tate buffers, fragmentation occurs rapidly, as expected (Scheme 3) [28]. The rate of decarboxylation is not affected as the concentration of buffer is increased. However, the extent of fragmentation relative to the formation of HBnTh decreases. In the reaction cata- lyzed by BFD, although the mechanism appears to require formation of the analogous carbanion, HBnThDP forms without competition from what should be a faster fragmentation [21,22,29–31]. This presents an interesting problem: how does the enzyme avoid fragmentation if that process without interven- tion of an enzyme has a lower barrier than the normal pathway of the enzyme [32,33]? Cryptic catalysis – decarboxylation of MTh is enhanced by pyridine acids Based on conventional analysis of the mechanism of decarboxylation of a thiamin conjugate, there is no role for a catalyst in the carbon–carbon bond-breaking Scheme 2. Fragmentation from the C2a conjugate base of 2-(1-hydroxybenzyl)thiamin is a very fast process. Scheme 3. Decarboxylation of MTh leads to fragmentation in the absence of an enzyme or Brønsted acid. R. Kluger and S. Rathgeber Catalyzing separation of carbon dioxide FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS 6091 step [5,34]. The thiazolium nitrogen is in the position that corresponds to the carbonyl oxygen in a 2-keto- acid. While an acid can protonate a ketone’s carbonyl oxygen, the thiazolium nitrogen is at its maximum electron deficiency and has no available coordination sites. Simply, there is no place for a proton or other cation to position itself in order to promote the reac- tion by stabilizing a transition state that resembles the product. This means that neither Brønsted nor Lewis acids can play a role in promoting cleavage of the carbon–carbon bond. Surprisingly, it struck us as being most remarkable when Hu and Kluger observed that pyridine buffers promote decarboxylation of MTh [35]. Their investiga- tions revealed that only the acid component of the buffer is catalytically active. In addition, C-alkyl substituted pyridine-derived acids also acted as cata- lysts, even those with alkyl substituents adjacent to the nitrogen center. However, no other acids or bases that were tested were effective [18]. The second-order rate constants for catalysis by the various pyridine deriva- tives are essentially invariant. The lack of dependence on pK a is not consistent with catalysis by Brønsted bases from a weaker acid substrate (Fig. 2). This sug- gests that the catalytic process is a thermodynamically favorable proton transfer [18]. The only site that becomes available for protonation in the decarboxylation reaction of MTh is C2a. This is accessible only after carbon dioxide has formed. Thus, in order for the pyridine acids to be catalytic, the for- mation of carbon dioxide would have to be reversible (Scheme 4). The role of the catalyst would be to add a proton to compete for the carbanion against carbon dioxide. This process slows the reverse reaction and in doing so accelerates the diffusional separation of carbon dioxide and HBnTh. An alternative possibility is that the charge of the protonated material provides electrostatic stabilization in the transition state for bond cleavage, without transferring a proton (Scheme 5). While this may be a generally applicable type of mechanism [36], it is unlikely to be in operation here as the proton’s position is necessarily dynamic – rapidly associating and dissociating. As a measure of the significance of the electrostatic effect, we added N-ethylpyridinium chloride and observed that it has no effect on the rate of reaction, and any electrostatic effect is therefore very small, if any [18]. Pre-association mechanisms Jencks [37] and Venkatasubban and Schowen [38] observed catalysis in reactions where separation of products from one another by diffusion is rate-deter- mining. They reasoned that in such a case, the catalyst must form an initial, stable complex with the reactant. Applying that concept to the present case, the pyridi- nium catalyst must be associated with MTh prior to 98 CH 3 CH 3 CH 3 CH 3 H 3 C H 3 C 76 pK a 54 –5 –4 –3 log K obs –2 –1 N H N H N H N H Fig. 2. Second-order rate constants for catalysis of the decarboxyl- ation of MTh by Brønsted acids derived from pyridine and C-alkyl- pyridines. The fitted line has a slope of 0, consistent with a thermodynamically favorable proton transfer. Scheme 4. The complex of protonated pyridine and MTh accelerates departure of carbon dioxide, trapping the carbanionic product as it forms. Catalyzing separation of carbon dioxide R. Kluger and S. Rathgeber 6092 FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS the decarboxylation process. In the reported instances [37,38], the pre-associated catalyst is held in place by hydrogen bonding, and is in a position to transfer a proton to the acceptor in competition with a reversible step. However, in the decarboxylation of MTh, there are no groups with which a proton donor could form a hydrogen bond to promote the reaction. This suggests that attractive forces in pre-association processes are not necessarily limited to hydrogen bonding. Based on modelling, using analogous materials as the basis, we proposed that pyridinium can be associated with MTh by face-to-face p-stacking interactions with aromatic groups of MTh [18]. This can position the proton- donating site near the carbanion that is being produced. As carbon dioxide forms, the associated protonated pyridine is in a position from which it can readily transfer a proton to the nascent carbanionic C2a posi- tion derived from HBnTh. The acid’s proton competes with carbon dioxide as an electrophile. Thus, the over- all protonation process facilitates the diffusional sepa- ration of carbon dioxide and HBnTh. This requires that we consider the possible existence of an additional intermediate that leads to the rate-determining step. The complex in which carbon dioxide remains associ- ated with the conjugate base of HBnTh must be dis- tinct from that in which carbon dioxide has separated. Decarboxylation as a two-step process Based on the idea of a pre-associated catalyst and the observed catalysis by pyridinium, we proposed that, in general, separation of carbon dioxide and the conju- gate base of HBnTh is at least partially rate-determin- ing [18]. If the barrier for addition of carbon dioxide to the newly formed carbanion is lower than the bar- rier to diffusional separation, then diffusion is neces- sarily the rate-determining step. Lowering the barrier for the diffusion step is therefore the only way to accelerate the reaction. As diffusion is the result of a set of physical properties, the process itself cannot be accelerated. Instead of affecting diffusion as a process, a reaction can proceed faster if the process competing with diffu- sion is slower. This is the case if the decarboxylation step is reversible. Analysis requires consideration of the relative magnitudes of the barriers for diffusion of carbon dioxide and reversal of decarboxylation (Scheme 6). Gao et al. calculated reaction pathways for the non- enzymic and enzymic decarboxylation of orotidine Scheme 5. Electrostatic stabilization of the transition state for decarboxylation of MTh. Scheme 6. The intermediate is associated with carbon dioxide. The lower barrier is associated with k )1 , and k 2 is the rate determining step. R. Kluger and S. Rathgeber Catalyzing separation of carbon dioxide FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS 6093 monophosphate [39,40]. Their calculations show that the enthalpic kinetic barrier to the reverse reaction, addition of the carbanion to carbon dioxide, is very small or non-existent. The barrier to the reaction is purely entropic, and arises once carbon dioxide and the residual anion are separated by solvent mole- cules. Applying this idea to decarboxylation reactions in general, diffusion of carbon dioxide can be the rate-limiting step in a two-step process where the barrier to reversion is lower than the barrier to dif- fusion. If the acid catalyst suppresses the reverse reaction, it will make the overall forward reaction faster. Facilitating rate-limiting CO 2 diffusion If there is no additional enthalpic barrier to addition of the carbanion to an associated molecule of carbon dioxide, the rate constant will be approximately the same as the frequency of vibration of a carbon–carbon bond. The stretching frequency of such a bond is typically approximately 1000 cm )1 , which corresponds to a rate constant of 3 · 10 13 s )1 . Carbon dioxide is internally polarized, with the elec- tron deficiency at carbon creating a partial positive charge relative to the electronegative oxygen atoms. The center of the molecule will be attracted to the relatively anionic C2a centre of the conjugate base of HBnTh, introducing a barrier to separation. (The oxygen atoms may be attracted to the cationic thia- zolium nitrogen as well.) We estimate that the rate constant for diffusional separation will be somewhat smaller than for cases where there is no attractive force, a maximum of approximately 10 8 s )1 . The ratio of recombination to separation based on these esti- mates is approximately 10 5 . If an enzyme efficiently promotes the separation process by direct protonation of the residual anion, the acceleration is approximated by this ratio. This ratio of 10 5 is about the same as that of between k cat for BFD to the unimolecular decarboxylation rate constant of MTh. If the enzyme achieves this by transferring a proton to the incipient carbanion, it will also completely suppress fragmenta- tion of the coenzyme [3,18]. Formation of a productive complex between proton- ated pyridine and MTh prior to decarboxylation allows proton transfer to compete with the capture of carbon dioxide by the nascent enamine. This provides a catalytic route for decarboxylation simply by provid- ing a competitor for the back reaction of carbon diox- ide, promoting a net reaction in the forward direction. As carbon dioxide does not have specific binding sites in a protein, the reaction becomes effectively irrevers- ible once it separates from its co-product after the enamine is protonated. Extension – enzymes always use pre-association to promote reactions Enzymes bind substrates into active sites that contain multiple functional groups in close proximity. While pre-association of two molecules in organic chemistry is an uncommon component of catalysis, it is a universal aspect of enzymic catalysis [41]. Therefore, non-enzymic reactions that involve pre-association provide information on key aspects of enzymic reac- tions. Although intramolecular reactions model reac- tions of bound substrates [42], the structural relationships of functional groups and geometric restrictions limit interpretations. The role of the protein We propose that the observed (spontaneous) first-order rate constant for decarboxylation of MTh is very small compared to the k cat of BFD because the enzyme con- tains pre-associated functional groups that can serve as the source of the proton necessary to block the return of carbon dioxide by protonating the nascent enamine [18]. This proposal explains why, even though the non- enzymic decarboxylation product fragments with a rate constant greater than the k cat for BFD, this is not an issue simply because the intermediate is protonated much more rapidly than it fragments. As fragmenta- tion occurs from the unprotonated intermediate, the enzyme catalyzes the reaction and at the same time blocks the destructive pathway, without further evolu- tion of function. Enzymic catalysis of carboxylation – the same issues in reverse In enzyme reactions, the pathways for carboxylation are not the reverse of those for decarboxylation because aspects of energy and equilibrium make the situation more complex. Many of the questions that we are addressing in terms of reversible formation of carbon dioxide during decarboxylation have been con- sidered in depth with respect to carboxylation reac- tions. In 1975, Sauers et al. speculated on the potential role of carboxy phosphate in the carboxylation of bio- tin [43]. They suggested that it is a source of localized carbon dioxide, which is the same situation as arises spontaneously in decarboxylation. They proposed that the hypothetical anhydride of carbonic and phosphoric acids, from the enzyme-catalyzed reaction of ATP and Catalyzing separation of carbon dioxide R. Kluger and S. Rathgeber 6094 FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS bicarbonate, would be too unstable to serve as an intermediate. Instead, it spontaneously converts (with a half life of less than a second) to carbon dioxide and phosphate. The resulting unsolvated carbon dioxide is then in a position to react with an adjacent bound nucleophile (Scheme 7): ‘The high local concentration of this molecule of carbon dioxide provides an effec- tive driving force for its reaction with the bound acceptor so long as it reacts with the acceptor more rapidly than it dissociates into solution. A molecule may have a high Gibbs free energy that makes it effec- tively an ‘energy-rich’ compound as a consequence of its fixation and decreased entropy, as well as chemical activation ’ [43]. Furthermore, Sauers et al. address the observation that the same enzyme facilitates the reverse process, the decarboxylation of N 1 ¢-carboxybiotin: ‘If the rate- determining step of this reaction is the dissociation of bound carbon dioxide, the addition of acceptor mole- cules that decrease the steady-state concentration of carbon dioxide at the active site would decrease the observed rate of decarboxylation. This is consistent with the observed inhibition of carboxybiotin break- down by inorganic phosphate’. Thus, they propose that carbon dioxide reacts with an alternative nucleo- phile that gives an unstable covalent intermediate, pre- venting it from reacting with the group that would reverse the reaction. This strategy for accelerating decarboxylation by facilitating the diffusion of carbon dioxide is complementary to one in which a proton is added to the residual organic anion. Despite the fine logic and elegance of this proposal, the idea of revers- ible decarboxylation as presented received little further attention. Catalysis by addition to carbon dioxide An alternative means of promoting the separation of carbon dioxide suggested in the paper by Sauers et al. [43] involves trapping the carbon dioxide with a com- peting nucleophile (Scheme 7, last step). A carbon dioxide-trapping mechanism, in which a nucleophile on the enzyme adds to carbon dioxide as it forms, might also be a relevant step in some ThDP-dependent decarboxylases (Scheme 8). There are indications of this possibility in the reaction of benzoylphosphonate as a substrate analog of benzoylformate in the reaction catalyzed by BFD. Benzoylphosphonate appears to be a mechanism-based inactivator: it is processed by the enzyme leading to a product that effectively inactivates the enzyme [44]. Crystallographic analysis reveals that the equivalent of metaphosphate is transferred to the hydroxyl group of an active site serine to give a phosphate monoester. If this occurred with the normal substrate, the metastable carbonate monoester would result. Trapping carbon dioxide temporarily would slow the reversal. At the same time, a proton would have been added in place of carbon dioxide at the basic reaction site, yielding the stable product. AHAS – does the flavin cofactor maintain reversibility? Acetohydroxy acid synthase (AHAS) catalyzes decar- boxylation of a 2-ketoacid conjugate of ThDP followed by addition of the enamine-carbanion to a second 2-keto- acid [45]. It is essential that the carbanion generated by decarboxylation is not protonated in order for the C2a center to function as a nucleophile. FAD is an essential Scheme 7. Formation of ‘low-entropy’ carbon dioxide and formation of carboxybiotin. R. Kluger and S. Rathgeber Catalyzing separation of carbon dioxide FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS 6095 cofactor for the enzyme, but no role for an oxidation process in catalysis has been discovered, although the flavin may be reduced slowly during the process of catal- ysis [45,46]. As AHAS resembles pyruvate oxidase, a functional flavoenzyme, it has been proposed that the flavin is only an evolutionary vestige in AHAS with a purely structural role [45,46]. Nonetheless, it is intrigu- ing to consider how the cofactor might function in a more active sense. It is reasonable to assume that evolu- tion would have favored an enzyme that did not require the complexity of the cofactor. In terms of the general mechanism we have pre- sented, the flavin could serve as a temporary storage site for the electrons liberated in the decarboxylation process, preventing addition of carbon dioxide and reversal of the reaction, or providing a proton to quench the intermediate in the absence of the second substrate under dilute reaction conditions (Scheme 9). It is well-known that redox cofactors in other enzymes can complete an oxidation–reduction cycle as a means of altering the reactivity of intermediates, giving the external appearance of an inactive cofactor [47–52]. The flavin is reduced during the course of catalysis, although a net reduction occurs only if the oxidized substrate decomposes through an independent uncata- lyzed pathway [45]. Implications from site-directed mutagenesis on benzoylformate decarboxylase The observation of pyridine acid-catalyzed decarboxyl- ation of MTh suggested that ThDP-dependent decar- boxylases utilize Brønsted acids in the active site to facilitate departure of carbon dioxide, protonating the C2a carbanion-enamine intermediate prior to separa- tion of carbon dioxide. This prevents reversal to form Scheme 8. A base-catalyzed reaction with the side chain of serine competes for carbon dioxide with the nascent carbanion of HBnThDP. Scheme 9. Proposal for a catalytic role for the flavin in AHAS. Catalyzing separation of carbon dioxide R. Kluger and S. Rathgeber 6096 FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS the carboxylic acid. Therefore, we expect that proton sources at the active site of the enzyme would facilitate the reaction. Polovnikova et al. reported the effects of site-direc- ted mutagenesis replacements of active site groups in BFD [53]: S26A, H70A and H281A. Their results indi- cated that catalysis of the bound substrate is most sub- stantially affected by H70A, for which k cat is reduced by a factor of over 1000, while the reduction in H281A was significant but smaller. However, the cata- lytic rate constants of H70A and H281A are still orders of magnitude higher than the rate constants for uncatalyzed decarboxylation of MTh [11,18]. Polovnik- ova et al. make the intriguing proposition in the abstract of their paper that protonation of the groups on the enzyme is distinct: ‘The residue H70 is impor- tant for the protonation of the 2-a-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-a-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine’. However, the main role proposed for H70 is as a Brønsted acid to proton- ate the carbonyl of the substrate to promote addition of the ylide of ThDP. On the other hand, if H70 is the most effective proton donor in suppressing the return of carbon dioxide, its replacement by alanine would lead to a structural change and the loss of one site from which a proton could be donated. However, as proton association is a dynamic and rapid process, catalysis is still highly effective as other groups serve as proton sources such that the k cat value remains well above the rate constant for the uncatalyzed decarbox- ylation of MTh, suggesting that rescue is possible by other groups [54]. Interpreting saturation mutagenesis In a recent report on BFD, Yep et al. [54] used satura- tion mutagenesis as a probe to determine the extent of decrease in activity as a result of substitutions for active site histidines H70 and H281. They report that H281F has a k cat value that is 20% of that of the native protein. This is consistent with other acid groups being able to take on its role but with lower efficiency. They also found that replacing H70 with threonine or leucine decreases the activity to approxi- mately 3% of that of the native protein, while the pre- viously reported substitution with alanine causes a reduction of k cat to 0.025% of the native level, indicat- ing that a more serious structural change occurs. As none of the substituted groups have a specific role to play as a catalyst, it is likely that the structural changes have varying effects on the arrangement of groups in the active site that promote the departure of carbon dioxide. Nonetheless, even the slowest mutant, H70A, is approximately 1000 times more reactive than MTh. Yep et al. [54] state that mutagenesis can thus be misleading in assigning mechanistic roles, but it is clear that the roles for the putative catalytic residues of BFD are not discreet, and it is difficult, if not impossible, to assign them definitive functions by any experimental means. Structural implications in facilitated carbon dioxide departure – evidence from sequence homology and saturation mutagenesis The relevance of specific amino acid residues can often be deduced from their conservation in enzymes from different species or in those sharing the same or similar mechanisms. Given that the mechanism of ThDP- dependent decarboxylation is similar regardless of the substitution pattern of the substrate, a significant amount of sequence homology should exist among decarboxylases if specific catalytic residues are neces- sary for catalysis. However, this is not the case [55]. Apart from the residues that bind ThDP in the active site, the structures of pyruvate decarboxylase and BFD are very different [23,54,56]. Thus, it is likely that the structural features of the protein that interact with the substrate also facilitate the decarboxylation process. This is consistent with the observation that the H281F mutant of BFD retains the greatest activity of the active site variants at this position despite the different functional groups in the side chains [54]. As steric bulk is a common structural feature of histi- dine and phenylalanine, the role of the amino acid at position 281 may be to influence the conformation of ThDP-bound intermediates. Conformational fluctua- tion of BFD intermediates in the active site is observed in ThDP carboligation, which generates 2-hydroxy- ketones [57]. The stereoselectivity of these reactions is a function of the size of the acyl donor bound by thia- min. Less differentiation was observed with small sub- stituents on the acyl donor, presumably due to a small difference in the kinetic barrier between conformations, leading to attack of either face of the acyl acceptor [57]. Conclusions ThDP-dependent enzymes catalyze remarkable reac- tions, providing catalysis well beyond that which would result from the cofactor alone. We have pro- posed that decarboxylation is enhanced by the ability of the protein to provide relatively acidic groups in R. Kluger and S. Rathgeber Catalyzing separation of carbon dioxide FEBS Journal 275 (2008) 6089–6100 ª 2008 The Authors Journal compilation ª 2008 FEBS 6097 proximity to the specific site of decarboxylation. The protein accommodates the substrate specifically and presents a resilient proton pool from sites that are con- siderably more acidic than solvent water. 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