Báo cáo khoa học: Catalyzing separation of carbon dioxide in thiamin diphosphate-promoted decarboxylation ppt

Catalyzing separation of carbon dioxide in thiamindiphosphate-promoted decarboxylation Ronald Kluger and Steven Rathgeber Davenport Chemical Laboratories, Department of Chemistry, Univer

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.

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 106 times

smaller than the typical kcatvalue 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 106 times

smaller than the kcatfor 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 105 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.

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

104s)1 at 30C, which is approximately 100 times

larger than the kcatof 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.

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 transireac-tion 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 pKa 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

9 8

CH 3

CH 3

CH 3

CH 3

H3C

H 3 C

7 6

pKa

5 4

–5

–4

–3

Kobs

–2

–1

N

N N

N

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.

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 non-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.

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

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· 1013s)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 108s)1 The ratio

of recombination to separation based on these

esti-mates is approximately 105 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 105 is about the same as

that of between kcat 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 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 kcatof 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 kcat 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 funcevolu-tion

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

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 N1¢-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.

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.

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 kcatis 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 substrproton-ate 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 kcatvalue 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 kcat 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 kcatto 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

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 While a

mutation that replaces a Brønsted acid with a

hydro-carbon lowers overall activity, the residual activity is

still much greater than that of the intermediate itself in

the absence of the protein These results suggest that

ThDP intermediates undergo facilitated reactions in

protein environments, and that there is sufficient

cata-lytic redundancy in the sources of protons for acid

catalysis to assist departure of carbon dioxide

Acknowledgements

We thank the Natural Sciences and Engineering

Research Council of Canada for support through a

Discovery Grant (R.K.) and a Postgraduate

Scholar-ship (S.R.)

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