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 ª2009 The Authors Journal compilation ª2009 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
CH
3
COCO
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 ª2009 The Authors Journal compilation ª2009 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+
SR2
HO
Ph
N
SR2
N
SR2
Ph
HO
Ph
HO
N
SR2
ylide
Mechanism of benzaldehyde lyase
–
C2α-carbanion/enamiine
+
R1
R1
+
–
k
2
HN
N
N
S
NH
R2
H
N
N
N
S
NH2H
+
4'-aminopyrimidinium
+
1',4'-iminopyrimidine
R2
N
N
N
S
NH2
4'-aminopyrimidine
+R2
thiazolium
–H1'
–H4'
1'
4' 2
3'
k–2
R1
R1
N+
SR2
Ph
OH
R1
+
H
k1/k–1
PhCHO
HBThDP
k–4
k4
k–5
k5
PhCHO
AP
APH+
IP
λmax380 nm
Ph Ph
O
OH
Ph OH
DDEThDP
PhCHO
PhCHO
k3
k–3
Ph = C6H5
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 ª2009 The Authors Journal compilation ª2009 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 aand bphosphoryl 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 ª2009 The Authors Journal compilation ª2009 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
C2aatom, 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 ª2009 The Authors Journal compilation ª2009 FEBS