Keywords 1¢,4¢-iminopyrimidine tautomeric form of thiamin; benzaldehyde lyase; benzoylformate decarboxylase; CD; enamine intermediate; pyruvate decarboxylase; pyruvate dehydrogenase; thi
Trang 1Reaction 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.
Trang 2This 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 pKa 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 funcsatura-tion 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/ C2 α-carbanion, AP(or APH +) +
+ R1
R1 + –
k2
k3
k 5
R1 = 4'-amino-2-methyl-5-pyrimidyl R2 = β-hydroxyethyldiphosphate
OH
S8-acetyldihydrolipoyl-E2
R2
CH3C OCO 2
-
CO 2
k –MM
R1
R1
R1
k 4
lipoyl-E2
2-AcThDP, AP (or APH +)
S S
E2
SH S
E2
CoASH
CH3COSCoA
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
NH2
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
Ktautomerization
H3COC
MM, AP
N
S
Me
R2
Me
HO H
+ R1
HEThDP, IP
k6
k –6
Scheme 2 Mechanism of E coli and human pyruvate dehydrogenase complex with role of ThDP.
Trang 3only led to a 30-fold penalty on kcat⁄ Km 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· 106
reduction in kcat, with little impact on kcat⁄ Km [20]
and (b) ketosteroid isomerase (EC 5.3.3.1), where
sub-stitution of the catalytic Asp38 by Asn leads to a
105.6 decrease in kcat [21], whereas substitution of the
same residue by Ala only reduced the kcat 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 pKa= 5.3 at the acidic side of either the kcat-pH or kcat⁄ Km-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 lackhydrogen-bond-ing 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 +
HO Ph
N
N
Ph HO
Ph HO
N
ylide
Mechanism of benzaldehyde lyase
– C2 α-carbanion/enamiine
+
R1
R1 + –
k 2
HN
N
N S NH
R2 H
N
N
N S
NH2 H
+
4'-aminopyrimidinium
+ 1',4'-iminopyrimidine
R2
N
N
N S
NH2
4'-aminopyrimidine
thiazolium –H1'
–H4'
1'
4' 2 3'
k–2
R1
R1
N +
Ph OH
R1
+
H
k1/k–1
PhCHO
HBThDP
k–4
k4
k–5
k5 PhCHO
AP
APH +
IP
λ max 380 nm
O
OH
DDEThDP
PhCHO
PhCHO
k3
k–3
Ph = C6H5
Scheme 3 Mechanism of benzaldehyde lyase.
Trang 4ThDP 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 Mg2+ or Ca2+, the divalent metal
serving as an anchor in a highly tailored environment
with a universally conserved GDG recognition site and the diphosphate-Mg2+ binding motif consisting
of a GDG-X26-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 D2O, 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 1012–1013 [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.
Trang 5from 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 pKa 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 ionizainforma-tion 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 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
Trang 6the 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 pKa 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 15N 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 pKafor 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
BFDC (pH > 7.0)
E51D YPDC-MAP YPDC-AcP -E571A E1ec-Py E401K E1ec-Py POX-AcP)
E51D YPDC-MAP YPDC-AcP) E1ec-MAP E1ec-AcP) E1h-AcP) POX-AcP) BAL-BF BAL-PPy BFDC-MBP BAL-MBP
YPDC+acetaldehyde
Trang 7AP, are quite distinct [45]; early 15N 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 pKa 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
pKa= 7.42 for the ([AP]+[IP])⁄ [APH+] equilibrium
on BAL (Fig 2) This pKa 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 pKa 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 pKa 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 Ktautomer, in conjunction with the pKa 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 pKa and
Ktautomer 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, pK1¢and pK4¢have similar mag-nitudes; the enzymes shifted the pK4¢ 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
1010s)1Æm)1 (giving a pK2 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 101–102
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 pKa, 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 + )
Trang 8bases 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 the13C 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 13C
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].
Trang 9according 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 mixresult-ing 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 kmax 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 kmax 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.
Trang 10be observed (due to its long kmax 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¢-13C2]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 13C 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
1
0
Time (s)
8
6
4
2
0
–2
–4
–6
330 nm
305 nm
8 6 4 2 0
0 1 2 3 4 5 6 7 8 9 10
360 Wavelength (nm)
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 k1= 4.44 ± 0.34 s)1and k2= 0.593 ± 0.064 s)1were calculated
[44].
Wavelength (nm)
400 450 500 550 600
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 70
0
2
4
6
8
10
12
14
16
18
[ES]
Enamine LThDP analogue
k2 = 0.507 + 0.002 s–1
k3 = 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).