Enhanced reactivity of Lys182 explains the limited efficacy of biogenic amines in preventing the inactivation of glucose-6-phosphate dehydrogenase by methylglyoxal

Article abstract of DOI:10.1016/j.bmc.2011.01.044

This study examines the inactivation of the enzyme glucose 6-phosphate dehydrogenase (G6PD) by methylglyoxal (MG) and the eventual protection exerted by endogenous amines. To determine the protective effect of amines, the rate constant of the reaction of MG with the amino group of N-α-acetyl-lysine, carnosine, spermine and spermidine was measured at pH 7.4, and the behavior of endogenous amines was analyzed on the basis of quantum chemical reactivity descriptors. A 63% reduction in the enzyme activity was found upon incubation of G6PD with MG at pH 7.4. The inactivation of G6PD was even larger when the pH was increased to 9.4, revealing a weak protective effect by the amines. The results suggest that some basic residues of G6PD exhibit an anomalous reactivity, which likely reflects a shift in the standard pK_a value due to the local environment in the enzyme. Under the experimental conditions used in the assays, this hypothesis was corroborated by mass spectrometry analysis, which points out that modification of Lys182 in the binding site is responsible for the inactivation of G6PD by MG. These results emphasize the need to search for more effective antiglycating agents, which can compete with basic amino acid residues possessing enhanced reactivity in proteins.

Full text of DOI:10.1016/j.bmc.2011.01.044

Bioorganic & Medicinal Chemistry 19 (2011) 1613–1622
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhanced reactivity of Lys182 explains the limited efficacy of biogenic amines
in preventing the inactivation of glucose-6-phosphate dehydrogenase by
methylglyoxal
Patricio Flores-Morales a, Claudio Diema b, Marta Vilaseca b, Joan Estelrich a, F. Javier Luque a,
Soledad Gutiérrez-Oliva c, Alejandro Toro-Labbé c, Eduardo Silva d,
⇑
a Departament de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain
b Unitat de Espectrometría de Masses, Institut de Recerca Biomèdica, Baldiri i Reixac 10, 08028 Barcelona, Spain
c Laboratorio de Química Teórica Computacional (QTC), Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 9840436, Chile
d Laboratorio de Química Biológica (QBUC), Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 9840436, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
This study examines the inactivation of the enzyme glucose 6-phosphate dehydrogenase (G6PD) by
methylglyoxal (MG) and the eventual protection exerted by endogenous amines. To determine the pro-
tective effect of amines, the rate constant of the reaction of MG with the amino group of N-a-acetyl-
Received 22 November 2010
Revised 14 January 2011
Accepted 21 January 2011
Available online 27 January 2011
lysine, carnosine, spermine and spermidine was measured at pH 7.4, and the behavior of endogenous
amines was analyzed on the basis of quantum chemical reactivity descriptors. A 63% reduction in the
enzyme activity was found upon incubation of G6PD with MG at pH 7.4. The inactivation of G6PD was
even larger when the pH was increased to 9.4, revealing a weak protective effect by the amines. The
results suggest that some basic residues of G6PD exhibit an anomalous reactivity, which likely reflects
a shift in the standard pKa value due to the local environment in the enzyme. Under the experimental
conditions used in the assays, this hypothesis was corroborated by mass spectrometry analysis, which
points out that modification of Lys182 in the binding site is responsible for the inactivation of G6PD
by MG. These results emphasize the need to search for more effective antiglycating agents, which can
compete with basic amino acid residues possessing enhanced reactivity in proteins.
Keywords:
Maillard reaction
Methylglyoxal glycation
Glucose 6-phosphate dehydrogenase
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
of amino groups in apolar internal sites can alter the pKa and in-
crease the population of the neutral form,5–7 thus making those
The Maillard reaction is a non-enzymatic glycation process that
involves the condensation of a carbonyl-containing molecule and a
free amine. In the early steps of glycation, carbonyl groups of sug-
ars react with amino groups of proteins through the formation of
Schiff bases, which can then lead to Amadori rearrangements1–4
(Fig. 1a). The efficiency of the imine generation depends on the
amount of amino groups in neutral form capable of nucleophilic at-
tack toward the carbonyl group. Although the neutral species
should be marginally populated at physiological pH, embedding
groups more reactive against glycating reagents.
Besides sugars, attention has also been directed toward a-dicar-
bonyl compounds such as glyoxal and methylglyoxal (MG), which
can react with amino, guanidinium and thiol groups of proteins.
They are active crosslinkers. In lens MG is formed from the degra-
dation of triose-phosphates,8 glycated proteins9 and lipid peroxi-
dation,10 reaching
a
relatively high content (ꢀ1–2
l
M)11
compared to blood samples of healthy human subjects (ca.
80 nM).12 The later stages of the complex reactive processes trig-
gered by
a-dicarbonyl compounds give rise to a variety of prod-
ucts, which are denoted as advanced glycation endproducts
(AGEs; Fig. 1a). Examples of non-crosslinking AGEs are hydroimi-
Abbreviations: MG, methylglyoxal; NLys, N-
a-acetyl-Lys; Car, L-carnosine; Sp,
spermine; Spe, spermidine; NH2-group, -amino group; AGEs, advanced glycation
e
e
end-products; G6PD, glucose 6-phosphate dehydrogenase; G6P, glucose 6-phos-
phate; NAD(P)+/NAD(P)H, oxidized/reduced nicotinamide adenine dinucleotide
(phosphate); FA, formic acid; LC–MS, liquid chromatography–mass spectrometry
analysis; PDB, protein data bank; MEAD, macroscopic electrostatics with atomic
detail.
dazolone adducts with arginine residues, N-
e-carboxymethyl or
N- -carboxyethyl lysines, and argpyrimidine. Crosslinking AGEs
e
involves heterocyclic structures such as MOLD (MG-derived lysine
dimmer; Fig. 1a) and GOLD (glyoxal-derived lysine dimmer;
Fig. 1a).
⇑
Corresponding author. Address: Departamento de Química Física, Facultad de
Química (536), Pontificia Universidad Católica de Chile, Casilla 306, Correo 22,
Santiago 9840436, Chile. Tel.: +56 2 3544395; fax: +56 2 3544740.
E-mail address: esilva@uc.cl (E. Silva).
Several studies have examined the therapeutic implications of
Maillard chemistry to prevent the formation of AGEs.13 Biogenic
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.044

1614
P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
Figure 1. (a) Schematic representation of the glycation reaction. An
can evolve to an Amadori product containing a new carbonyl unit able to react with another
leads to advanced glycation end-products (AGEs) and cross-linked proteins (MOLD, methylglyoxal-derived lysine dimer; GOLD, glyoxal-derived lysine dimer). (b) Chemical
structure of methylglyoxal, spermine, spermidine, carnosine and N- -acetyl-lysine.
e-amino group of protein 1 reacts with a carbonyl group of a reducing sugar forming a Schiff base, which
e
-amino group from the same or a distinct protein. In this latter case glycation
a
amines, such as spermine and spermidine (Fig. 1b), have received
much interest due to their ability to inhibit protein alteration
due to glycation in vitro.14 Carnosine (Fig. 1b) also seems to atten-
uate the formation of AGEs15 presumably by sequestering glyoxal
and MG,16,17 which can promote protein cross-linking between ly-
sines.18–20 Intense efforts have been dedicated to the discovery of
novel and safer antiglycating agents, such as aminoguanidine, urea
derivatives and diaminopropionic acids.14,21,22
Protein glycation is important in the pathophysiology of many
processes related with aging, especially in diabetics, where sugar
concentration in the body is unusually high. A well known example
is the accumulation of modified proteins triggered by age- and
environmental-related damage in eye lens, which is associated
with lens opacification and senile cataract.23 There is no removal
of lens proteins with age and they remain in an environment con-
taining reactive small molecules that can react with proteins
in vivo.24–27 Thus, it is known that glycosylation of lens proteins
is increased in human cataract lenses from diabetic patients.26–28
The glycated proteins become yellow fluorescent and aggregated,
as generally found in human cataracts.27,28
The biochemical characteristics of the eye lens include a rela-
tively high content of glutathione (GSH) in its reduced form
(10 mM),29 which protects against oxidative damage. Oxidation
transforms GSH to the disulfide form, which is reconverted via glu-
tathione reductase reaction. The NADPH required by this enzyme is
produced in the phosphate pentose pathway, where glucose 6-
phosphate dehydrogenase (G6PD) plays a key regulatory role. De-
creased levels of GSH found in human cataract lenses30,31 could re-
flect a low content of NADPH due to a diminished activity of G6PD.
In fact, a decreased activity of G6PD has been reported in cataract32
and in aged lens.33
To the best of our knowledge, the effect of MG on G6PD has not
been previously studied. The aim of this work is to identify the
molecular basis of the loss of G6PD activity promoted upon incuba-

P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
1615
tion by MG. To this end, we examine the protective effect of the
amino groups present in carnosine (Car), spermine (Sp) and sper-
midine (Spe) against the inactivation of G6PD by MG. Both exper-
imental and theoretical studies are performed to gain insight into
the residues implicated in the chemical modification promoted
by MG. In turn, this information is discussed in light of the catalytic
mechanism of G6PD.
G6PD]) and Arg ([Guanidinium-G6PD]) residues of G6PD,
respectively.
If we assume that (i) Lys and Arg residues in the enzyme retain
’
the standard pKa s of the free residues, (ii) the rate constant of ly-
sines is given by the value determined for N-a-acetyl-lysine
(1.76 ꢁ 105 Mꢂ1 hꢂ1), and (iii) the rate constant of arginines can
be estimated from the ratio between the reaction rates of MG with
hippuryllysine35 and N-
a
-acetyl-
L
-arginine36 (1.21 ꢁ 106 Mꢂ1 hꢂ1),
then the FNH amine values calculated for Car, Sp and Spe at pH 7.4 are
2. Results and Discussion
2
0.92, 0.94 and 0.94, respectively. This indicates that around 7% of
MG reacts with basic groups present in G6PD. Accordingly, the
amount of Arg and Lys residues altered by MG should be extremely
low and could not explain the observed G6PD inactivation. In turn,
these findings suggest an augmented reactivity of some Arg and/or
Lys residues.
In order to increase the nucleophilicity of Car, Sp and Spe in the
reaction medium, the inactivation of G6PD was examined at higher
pH values. Remarkably, control assays performed in the absence of
MG showed that the enzyme activity remained unaltered after
16 h incubation (ez; Fig. 2), which rules out pH-induced structural
alterations in the G6PD activity. In the presence of MG the enzyme
activity is especially affected by the increase in pH from 7.4 to 8.4
(ez/MG; Fig. 2), which can be attributed to a more extensive mod-
ification of reactive basic residues. An improvement in the relative
protection by amines was also observed when the pH was in-
creased from 7.4 to 9.4 (Fig. 2). However, this effect is relatively
weak considering the high concentration of the amines.
The preceding findings allow us to hypothesize the existence of
basic residues in G6PD that are especially reactive toward MG
(Fig. S2 in Supplementary data). Among arginines, four potential
candidates for modification by MG are Arg46, Arg121, Arg223
and Arg395. Arg46 assists coenzyme binding through interaction
with the 20-phosphate of NADP+. Arg121 and Arg223 are close to
the glucose and adenine moieties of G6P and NADP+, respectively.
Finally, Arg395 forms a salt bridge that stabilizes the dimeric state
of the enzyme. Since MG incubations were made in the absence of
both substrate and coenzyme, Arg46, Arg121 and Arg223 are ex-
pected to be fully exposed to the solvent. Moreover, since the en-
zyme is in its monomeric state during incubation, Arg395 must
also be solvent-exposed. Under these conditions, the pKa of these
latter residues should be close to the standard one.
2.1. Determination of rate constants
The rate constants (k) for the reaction of MG with NLys, Car, Sp
and Spe at pH 7.4 were determined experimentally by the fluores-
camine assay.34 The rate constants range from 1.37 ꢁ 104
( 3 ꢁ 102) to 1.76 ꢁ 105 ( 5 ꢁ 102) Mꢂ1 hꢂ1 (Table 1). Sp and Spe
have very similar values, both being slightly more reactive than
Car, whose rate constant is around 10-fold smaller than that
derived for NLys. The differences between the rate constants can
be explained by the dual reactivity descriptor (Df(r); Eq. (3), see
Section 4) determined for representative structures of the amines
(See Fig. S1 in Supplementary data). Thus, whereas the NHþ3 -group
of Car has a large electrophilic lobule and deprotonation does not
increase its nucleophilic power, which instead concentrates in
the imidazole ring, deprotonation of Sp and Spe gives rise to a
notable increase in the nucleophilic power, which mimics the
trends found for NLys. In fact, the latter compound exhibits the
largest nucleophilicity. Thus, the distinct features of the dual
descriptor extension correlate well with the ordering of k values
for the amines (kCar < kSp < kSpe < kNLys; see Table 1).
2.2. G6PD inactivation mediated by MG
The effect of MG on the activity of G6PD was examined by incu-
bating the enzyme with MG in the absence of light for 16 h (pH
7.4). When the concentration of MG was 10-fold larger than that
of the lysine residues in the enzyme, a 63% reduction in the
G6PD activity was found (ez/MG; Fig. 2). Nevertheless, addition
of Car, Sp and Spe to the incubation mixture (ez/MG/Car, ez/MG/
Sp and ez/MG/Spe; Fig. 2) led to a moderate protection against
G6PD inactivation. The magnitude of the protective effect roughly
reflects the differences reported in Table 1.
Lys and Arg residues are sites susceptible to chemical modifica-
tion by MG.35,36 The fraction of NH2-groups of the protective
amines that might react with MG in the presence of G6PD can then
be estimated as:
In order to explore the ionization state of these residues, we
first carried out a series of PROPKA37,38 calculations for different
G6PD structures available in the PDB (see Section 4). PROPKA is
an empirical method for the structure-based prediction of the
pKa values of ionizable residues in proteins, which takes into ac-
kNH
½NH2-amineꢃ
2amine
FNH
¼
ð1Þ
2amine
kNH
½NH2-amineꢃ þ ke
½ NH2-G6PDꢃ þ kGuanidinium ½Guanidinium-G6PDꢃ
e
amine
NH2
2
where kNH
stand for the reaction rate of MG with the
count both intra-protein interactions and desolvation effects
through an empirically adjusted function related to the chemical
nature and spatial location of the residues. With regard to lysines,
there are five residues (Lys21, Lys148, Lys182, Lys338 and Lys343)
in the substrate binding site. According to PROPKA computations,
notable deviations from the standard pKa value (10.5) are found
for certain residues depending on the specific X-ray structure con-
sidered in the calculations. In particular, Lys148 and Lys182 are
predicted to have pKa values diminished by 1–2 units. This pKa
reduction is not unexpected taking into account an extreme exam-
ple of pKa value of 5.6 recently reported for a buried Lys in the
hydrophobic core of staphylococcal nuclease.7 To further check
these findings, additional computations were performed with
2ꢂamine
NH2-groups of neutral amines ([NH2-amine]), while ke
kGuanidinum denote the reaction rate with neutral Lys ([
and
NH2
e
NH2-
Table 1
Rate constants (k; Mꢂ1 hꢂ1) for the reaction between methylglyoxal
and several amines determined at physiological pH
Amine
k
Carnosine
Spermine
Spermidine
1.37 ꢁ 104 3 ꢁ 102
3.91 ꢁ 104 2 ꢁ 102
4.28 ꢁ 104 3 ꢁ 102
1.76 ꢁ 105 5 ꢁ 102
N-a-Acetyl-lysine

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P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
Figure 2. Inactivation of G6PD by MG and protective effect of amines. The reaction was carried out at 37 °C, pH 7.4–9.4 and dark conditions (ez: lysine residues + amino-
terminal group ratio into the enzyme; MG: methylglyoxal ratio; Car, Sp or Spe: carnosine, spermine or spermidine NH2-group ratio).
MEAD (Macroscopic Electrostatics with Atomic Detail).39 MEAD is
a method that relies on the detailed structural information of
atoms in a protein and applies a macroscopic dielectric model to
evaluate the solvent-screened electrostatic interactions in a sol-
vated macromolecular system. To this end, the protein is treated
as a low dielectric medium, characterized by the distribution of
charges located at atomic positions, immersed in a high dielectric
medium for the surrounding solvent, and the electric potential is
determined by solving the Poisson–Boltzmann equation. MEAD
calculations also showed a large dependence on the predicted de-
crease in pKa with the dielectric constant for the more buried Lys
residues (Fig. 3). This behavior is especially notable for Lys182
and Lys343, which are localized at the inner part of the binding
site, in comparison with Lys19 (totally exposed to the aqueous sol-
vent). Experimental evidence from the reaction rate of NLys with
MG at low water content and high proportion of dioxane, confirms
this fact (Fig. 3, insert).
2.3. Intact G6PD MS analysis
Intact proteins (native, MG-treated and NaBH4-reduced
MG-treated) eluted at a retention time of 31.18–32.05 min by
Figure 3. Reciprocal of calculated pKa for lysine residues (-j- Lys19, -d- Lys21, -h- Lys148, -s- Lys182, -4- Lys338, -N- Lys343) in the G6P binding site of a mutant of G6PD
(from Leuconostoc mesenteroides) as a function of the dielectric permittivity. Lys-19 is totally exposed to the solvent. [Insert: Variation of log krel (in Mꢂ1 hꢂ1) as a function of
1,4-dioxane content (by volume), for the reaction between NLys and MG].

P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
1617
Figure 4. Intact MS analysis of MG-treated and NaBH4-reduced MG-treated G6PD samples: (top) m/z ion distribution spectra and (bottom) Mass deconvolution spectra.
LC-nanoESI-MS coupling. An average mass of 54,440 Da, matching
the expected mass for G6PD, was observed as the main peak after
deconvolution of the MS spectra for the native and MG-treated
protein samples (Fig. 4; note that the mass includes also a methi-
onine residue at the N-terminal side in the protein used in MS
analysis). Deconvolution of the MS spectra recorded for the re-
duced sample affords a main peak with an average mass of
54,455 Da. Both in the non-reduced MG-treated sample and in
the NaBH4-reduced one, an additional mass, shifting +54–69 amu,
was also observed, which would agree with the formation of a
Schiff base on a basic residue. The higher proportion of this adja-
cent mass in the reduced sample versus the non-reduced one can
be explained by the higher stability of the hypothetical modifica-
tion after its selective reduction with NaBH4. Nevertheless, the
MS analysis of the intact protein is still not resolutely enough to
determine accurately the mass of the modified protein due to the
limitations of the deconvolution algorithm at high molecular
masses and in the presence of mixtures.
could be obtained using this strategy (Fig. S3 in Supplementary
data). Therefore, further protease digestion of the samples was per-
formed in order to localize precisely the main modification de-
tected on the protein sequence.
Peptides obtained from the protein digestion with the Glu-C
endoproteinase covered 83% of the protein sequence (Fig. S4 in
Supplementary data). For the MG-treated protein, two peptides
with monoisotopic masses of 2194.04443 and 2248.05932 were
found matching the monoisotopic masses of the peptide corre-
sponding to the sequence 166–183 (NAFDDNQLFRIDHYLGKE) with
no chemical modification and with a Schiff base modification of
Lys182, respectively (Fig. 5; see also Fig. S4). The isotopic distribu-
tions of the corresponding charged precursor ions perfectly
matched the theoretical isotopic distributions expected for both
peptides (see Fig. S5 in Supplementary data). The collected LC frac-
tion containing the eluted modified peptide was infused for high
resolution MS/MS analysis. Precursor ion (m/z 750.36080; Fig. 5,
top) was isolated and 37% energy was applied for CID fragmenta-
tion. The resulting fragments were deconvoluted to charge zero
and further analyzed with PROSIGHT PC software. Detection of one
MS/MS fragment ion of peptide 166–183 with less than 10 ppm
In order to make a more accurate mass determination of the
modified protein, the intact MG-treated G6PD sample was
reanalyzed on a 12 Tesla LTQ-FT mass spectrometer and data were
processed using ‘in-house’ deconvolution software (On-line auto-
mation CRAWLER software). A mass of 54463.16 Da was obtained
for the modified protein, differing only by two units from the
accuracy is indicative of the presence of
a modification
(+54.01001, Schiff base) in the C-terminal end of the peptide,
which can be attributed to the only Lys present in the peptide
(Lys182).
expected monoisotopic mass for
a Schiff base modification
(54461.14 Da). Nozzle-skimmer dissociation of the infused sample
resulted in the characterization of the C- and N-terminal ends of
the protein. However, no information about the core of the protein
The LC-nanoESI-MS analysis of the Glu-C digested NaBH4-re-
duced MG-treated protein also showed two peptides with monoi-
sotopic experimental masses of 1746.86893 and 1802.90990

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P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
Figure 5. LC-nanoESI-MS analysis of the Glu-C crude reaction of MG-treated and NaBH4-reduced MG-treated G6PD samples. Top: zoom spectrum at ion m/z 750.36080 (z = 3)
corresponding to peptide 166–183 from MG-treated G6PD and 902.46223 (z = 2) corresponding to peptide 170-183 from NaBH4-reduced MG-treated G6PD. Top Insert:
Xtracted chromatogram showing experimental retention time (25.5 and 24.4 min for m/z ions 750.36080 and 902.46223, respectively), bottom: Xtract deconvolution
spectrum of ions to the zero charged monoisotopic mass (Legend; RA, relative abundance; tr, retention time).
(Fig. 5; see also Fig. S4), which match the monoisotopic masses of
the expected G6PD Glu-C digested peptides corresponding to the
sequence 170–183 (DNQLFRHIDHYLGKE) in both non-modified
and reduced Schiff base forms, respectively. Further identification
was performed by comparison of the isotopic distribution of the
double charged experimental ion detected (m/z 902.46223; Fig. 5,
top) with the theoretical one considering the presence of a reduced
Schiff base in the peptide (see Fig. S6 in Supplementary data). On
the other hand, no matching was observed with the theoretical iso-
topic distribution of the same peptide considering a modification
by bi-formylation. After LC fraction collection, this peptide was in-
fused for MS/MS analysis. Following precursor ion (m/z 902.46223)
isolation, 40% CID energy was applied for fragmentation. Data anal-
ysis with Prosight PC software showed the presence of two MS/MS
fragment ions matching accurately (<10 ppm) a modification
(+56.02567, reduced Schiff base) in the C-terminal end of the pep-
tide, which can be attributed to chemical modification of Lys182.
Overall, these results indicate that, under the experimental con-
ditions used for the inactivation of G6PD with MG, the reduction in
the enzymatic activity can be attributed to the formation of a Schiff
base with Lys182. The enhanced reactivity of this residue in the
binding site would also explain the limited protection observed
upon addition of amines at physiological pH.
pKa. This suggestion is supported by distinct experimental
evidences.
(i) The plot of log kcat/Km versus pH reveals the presence of four
ionizable groups in Leuconostoc mesenteroides G6PD.40,41 In partic-
ular, two pKa values at 8.9 and 10.0 were assigned to amino acids
that affect the binding of G6P. Cosgrove et al. pointed out that res-
idues Lys182 and Lys343, which are located in the binding site that
accommodates the phosphate moiety of G6P, could be probable
candidates for those ionizations.41 Our results support the assign-
ment of the lowest pKa (8.9) to Lys182, whose enhanced reactivity
is reflected in the chemical modification by MG. In contrast, the
highest pKa (10.0) would correspond to Lys343.
(ii) These assignments are reinforced from the change in the
apparent Km for G6P (114 lM for the wild type enzyme in presence
of NADP at pH 7.6) induced by mutations of those basic residues.42
Mutation of Lys182 to either Gln or Arg gives rise to similar
increases in the Km value (Lys182?Gln: 13,100
16,100 M). In contrast, whereas the mutation of Lys343 to
Arg promotes a smaller change in the apparent Km (3080 M),
the mutation to Gln increases dramatically the Km value
(106,000 M). Therefore, the presence of a positive charge in posi-
lM; Lys182?Arg:
l
l
l
tion 343 is necessary for the binding of G6P, whereas there is great-
er flexibility regarding position 182. Accordingly, the highest pKa
value should be assigned to Lys343, enabling an Arg residue to play
a similar role. In contrast, the lowest pKa value would correspond
to Lys182, where mutation to either polar (Gln) or charged (Arg)
residues triggers similar shifts the Km value.
2.4. Ionization of Lys182 and its enhanced reactivity toward MG
The chemical modification of Lys182 by carbonyl groups reveals
an enhanced reactivity, which might arise from an anomalous low
Taken together, the preceding discussion allows us to conclude
that the enhanced reactivity of Lys182 is due to an unusual low pKa

P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
1619
value of 8.9, which facilitates the chemical modification by car-
bonyl groups of MG.
For instance, the addition of a second amine unit in gamma posi-
tion reduces the experimental pKa of propylamine from 10.6 to
8.6 (see Table S1 in Supplementary data). This factor likely contrib-
utes to the enhanced protective effect recently reported for N-ter-
minal 2,3-diaminopropionic acid peptides by Sasaki et al. who have
considered the use of 1,2-vicinal diaminoalkyl compounds as po-
From a biochemical point of view an important question is then
why Lys182 exhibits such a significant reduction in its pKa. We
hypothesize that the two Lys residues play distinct roles in medi-
ating the binding of G6P. Lys343 is directly implicated in the bind-
ing of the substrate through electrostatic stabilization due to the
formation of a salt bridge with the phosphate moiety of G6P. Nev-
ertheless, Lys182 might play an indirect role, as this residue could
participate in the protonation of His178 upon binding of G6P to the
enzyme. Inspection of the X-ray structures available for substrate-
bound forms of the enzyme (PDB entries 1E77 and 1E7Y; crystal-
lized at pH ꢀ7.6) indicates that the phosphate moiety of G6P is
tightly bound to both Lys343 and His178 (interatomic distances
of 2.6–2.8 Å). In fact, the relevance of His178 in binding G6P has
been confirmed by site-directed mutagenesis, as the His178?Asn
tential scavengers of a
-dicarbonyl compounds.48
The preceding considerations suffice to demonstrate that a
proper exploration of chemical substituents could be effective to
open new avenues for developing anti-glycating amines. Those
modifications must be properly chosen to yield an optimal pKa
that ensures a balance between ionization properties of the amine
and its intrinsic nucleophilicity. Thus, the reduction in the pKa
must be large enough in order to ensure the presence of signifi-
cant populations of the neutral species at physiological pH. How-
ever, as the intrinsic nucleophilicity due to the lone pair of the
deprotonated amine is also affected by the presence of the elec-
tron withdrawing atoms, those chemical substituents should be
carefully chosen as to retain the intrinsic nucleophilicity of the
neutral amine against carbonyl compounds. Thus, the precise nat-
ure and location of the chemical modifications made in the amine
should be properly chosen in order to keep the balance between
those factors.
Besides the direct chemical modification of the amine, an alter-
native approach for tuning the reactivity of amines might rely on
the use of microheterogeneous systems. The inclusion of amine
compounds into cyclodextrins or nanoparticles can provide a more
hydrophobic environment that enhances the nucleophilicity of the
amines. Thus, previous studies have shown that complexation of a
tertiary amine with b-cyclodextrin decreases the pKa (from 9.4 in
aqueous solution to 8.9 in 0.01 M b-cyclodextrin solution).49 Simi-
larly, the enhanced chemical reactivity of amines is also supported
by kinetic studies on the aminolysis of esters by primary alkyl-
amines in the absence and presence of cyclodextrins.50 Enhanced
deacylation of esters containing t-butylphenyl groups has also
been reported for systems comprising b-cyclodextrin linked to
dendrimer poly-ethylenimines.51
mutant showed
a 400-fold increase in the Km for G6P
(42,900
l
M).43 We propose here that the interaction of His178
with G6P is facilitated by the proton transfer from Lys182, thus en-
abling His178 to reorient its side chain to stabilize electrostatically,
in conjunction with Lys343, the binding of the phosphate moiety of
G6P.
From a therapeutic point of view the unusual reactivity of
Lys182 toward carbonyl groups explains the extremely low protec-
tion exerted by biogenic amines localized in the bulk of the
solution, notwithstanding the fact that they were used at concen-
trations 10-fold higher than that of the basic residues in the pro-
tein. This finding is in agreement with previous studies focused
on the structural requirements of a-dicarbonyl compounds impli-
cated in protein crosslinking,44 which have challenged the use of
compounds containing amino groups as a suitable strategy to
intervene against the Maillard reaction. Similarly, the local envi-
ronment of basic residues located in the interface of multimeric
aggregates, such as those found in the eye lens proteins, could also
explain the low protective effect of biogenic amines toward glycat-
ing agents.45,46
Taking into account that trapping of reactive dicarbonyl species
by drugs containing amine groups in their structures is one of the
strategies proposed to inhibit AGE formation, our results suggest
that the development of more effective therapeutic solutions to
the problem of glycation requires biogenic amines with increased
nucleophilicity, whose reactivity could then make an effective
competition with basic residues with anomalously low pKa values,
and therefore prone to chemical modification by carbonyl-contain-
ing compounds. In fact, the enhanced reactivity of those basic res-
idues likely contributes to the limited success of current Maillard
inhibitors, which justifies the intense research effort devoted to
the development of novel glycation inhibitors.47
Overall, the design of systems containing chemically modified
amines enclosed in less polar environments could be a promising
strategy to modulate the anti-glycating activity, thus enabling
the use of lower concentrations of amines. This latter aspect is also
relevant, taking into account the convenience to avoid side reac-
tions that can occur between amines and endogenous carbonyl
groups present in biological systems in vivo.52
3. Conclusions
The results reported here reveal that biogenic amines exert a
very modest protective effect on the inactivation of G6PD by MG.
This finding could be attributed to an anomalous reactivity of spe-
cific basic residues in the enzyme, which would then lead to an en-
hanced reactivity toward carbonyl groups. In the case of G6PD, the
results point out that, under the experimental conditions exam-
ined here, Lys182 should have an enhanced nucleophilicity, as this
residue is involved in the formation of a Schiff base modification by
MG. In fact, this residue is suggested to be responsible for the pKa
value of 8.9 determined experimentally in previous kinetic stud-
ies.41–43 Since Lys182 is found in the inner part of the binding site,
both limited exposure to water molecules and the nature of the
surrounding residues in the binding pocket, particularly His178,
could account for the enhanced nucleophilicity. In this context,
the design of therapeutic strategies that combine the modulation
of the intrinsic reactivity of amines through chemical modification
with the use of microencapsulating systems could be valuable for
developing more effective anti-glycating agents.
A plausible strategy to make more protective agents consists of
the introduction of suitable chemical modifications that enhance
the reactivity properties of amines. The effective tuning of the reac-
tivity of amines via chemical modification is supported by the
changes of pKa’s of aliphatic amines upon fluorination of the
methylenic chain (see Table S1 in Supplementary data). The pKa
of aliphatic amines is insensitive to the length of the chain, as
the pKa is close to 10.6 for methyl-, ethyl-, propyl- and butylamine.
In contrast, the inclusion of two fluorine atoms in beta position
reduces the pKa to 7.5. The addition of a third fluorine atom
(2,2,2-trifluoroethylamine) further reduces the pKa to 5.7. Clearly,
this effect can be attributed to the electron withdrawing effect of
fluorine, which facilitates deprotonation of the protonated amine.
Similar trends can also be observed in the pKa values predicted
for fluorinated analogs of alanine, as replacement of the methyl
group in the side chain of this residue by a trifluoromethyl unit
reduces the estimated pKa from 9.7 to 7.0 (see Table S2 in Supple-
mentary data). Other functional groups can trigger similar changes.

1620
P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
Two samples of G6PD incubated with MG at pH 8.4 under the
4. Materials and methods
4.1. Chemicals
same experimental protocol (16 h incubation, 37 °C, and absence
of light) were taken for mass spectrometry analysis. After incuba-
tion, NaBH4 was added to one of those samples in order to reduce
the Schiff base that might be formed upon modification of amino
groups by MG. Finally, a third sample of G6PD treated with the
same experimental protocol but without addition of MG was used
as control. The samples were then dialyzed for 6 days, and diluted
MG, Car, Sp, Spe, NLys, fluorescamine, G6PD, the oxidized form
of nicotinamide adenine dinucleotide (NAD+), glucose 6-phosphate
(G6P), were purchased from Sigma–Aldrich. Sodium borohydride
(NaBH4) was purchased from Fluka. Magnesium chloride (MgCl2)
was purchased from Merck. Acetonitrile (LC–MS quality, Scharlau),
water (LC–MS quality, Sigma–Aldrich), formic acid (FA) (LC–MS
quality, Sigma–Aldrich) and endoproteinase Glu-C (Roche) were
employed for liquid chromatography–mass spectrometry analysis
(LC–MS).
in ammonium acetate (25 mM) to obtain a final 10 lM concentra-
tion of the enzyme for intact MS analysis (top-down MS)55–58 or
digestion with Glu-C previous to MS analysis (middle-down
MS).59 In the latter case, a volume of 16
lL of endoproteinase
Glu-C (1 lg/lL in H2O) was added to 100 lL of protein solutions,
and incubation was performed at room temperature overnight
with gentle agitation. The samples were lyophilized and reconsti-
tuted in 50 lL of a 1% formic acid (FA) aqueous solution.
4.2. Determination of rate constants
Solutions of MG and amines were prepared in phosphate buffer
(pH 7.4) at a 100 mM concentration and incubated at 45 °C in the
absence of light. The decrease of the concentration of primary ami-
no group over time was followed by the fluorescamine assay.34 To
this end, assays were performed using a final concentration of
1 mM for NLys, Car, Sp and Spe, while the MG concentration was
varied from 25 to 100 mM when incubated with NLys or Car, and
from 50 to 200 mM when incubated with Sp or Spe. The reaction
rate is given by,
4.4. Intact protein MS analysis
For the top-down MS, an aliquot of 100 lL of the MG-treated
sample solution was cleaned-up and concentrated using a C4 zip-
tip (Millipore). Final elution from the zip-tip was made with 5 L of
a 78% acetonitrile, 1% acetic acid aqueous solution, and the sample
was further diluted in 10 L of an electrospray solution (methanol/
l
l
1% FA) for MS infusion analysis on a LTQ-FT 12 Tesla (ThermoFisher
Scientific). Direct sample introduction was performed by auto-
mated nanoelectrospraying using the Advion Triversa Nanomate
(Advion Biosciences) at 1.65 kV spray voltage and 0.6 psi gas pres-
sure. The mass spectrometer was operated on the positive polarity
mode and parameters were set to 200 °C capillary temperature,
42 V capillary voltage and 135 V tube lens. Full MS spectra (m/z
200–2000) were acquired at sid (source induced dissociation or
nozzle-skimmer dissociation) energies of 0 and 75 V and 200,000
resolution.
d½R-NH2ꢃ
ꢂ
¼ k½MGꢃ½R-NH2ꢃ
ð2Þ
dt
where ½R-NH2ꢃ and [MG] stand for the concentration of the primary
amino group and MG, respectively, and k is the actual rate constant.
Upon excess of MG, the concentration of the amino group is gi-
ven by [R-NH2] = [{R-NH2}]o eꢂkobst, where [R-NH2]o is the concen-
tration of the primary amino groups at t = 0, and kobs is the
observed or apparent rate constant.
A plot of ln ½R-NH2ꢃ=½R-NH2ꢃo versus time leads to a straight line
with slope ꢂkobs, and the representation of kobs against MG concen-
tration affords a linear plot with slope krel. Since the concentration
of non-hydrated MG, which is more reactive than the hydrated ad-
ducts, corresponds to 1% of the total MG present in the reaction
medium,53 the actual rate constant (k) is obtained from the expres-
sion krel ¼ k ꢄ FN; where FN corresponds to the fraction of amine
groups in their neutral nucleophilic form.
A volume of 10 lL of the 10 lM ammonium acetate reconsti-
tuted enzymes (native G6PD, treated and non-treated with MG)
was analyzed by on-line LC-nanoESI-MS. Samples were injected
through a Finnigan Micro AS autosampler (ThermoElectron Corpo-
ration) and loaded onto a BioSuite pPhenyl 1000 column (10 lm,
2.0 ꢁ 75 mm, Waters) using a Surveyor Finnigan MS quaternary
pump (ThermoElectron Corporation). A 60 min gradient of 5–80%
buffer B (A = 0.1% FA in water, B = 0.1% FA in acetonitrile) at
100 lL/min flow rate was used. The column outlet was directly
connected to an Advion Triversa nanomate (Advion Biosciences)
fitted to an LTQ-FT Ultra mass spectrometer (ThermoFisher Scien-
tific). The nanomate was used as a splitter (1:250) and as a nanoESI
source (Spray voltage and delivery pressure were set to 1.75 kV
and 0.3 psi, respectively). The mass spectrometer was operated in
positive polarity mode. Capillary voltage, tube lens and capillary
temperature were tuned to 35 V, 135 V and 200 °C, respectively.
Full scan MS spectra (m/z 295–2000) were acquired at 100,000 res-
olution (after accumulation of a target value of 106).
4.3. G6PD inactivation
The activity of G6PD was measured in a Genesys 10-S UV Scan-
ning Thermo Spectronic spectrophotometer using Langdom modi-
fied method.54 The total reaction mixture (3 mL) consisted of 2 mL
of a solution containing 1.84 U of G6PD (from L. mesenteroides) in
0.55 mM NAD+ and 1 mL of 1:1 mixture of MgCl2 (6.7 mM) and
G6P (1 mM). The activity was estimated from the increase in the
absorption band at 340 nm (corresponding to the formation of re-
duced nicotinamide adenine dinucleotide; NADH) for 1 min. Mea-
surements were performed in triplicate and the results expressed
as percentage of the activity measured at zero time incubation.
For the incubation of G6PD with MG and amines, a solution con-
4.5. Glu-C digested protein MS analysis
For the middle-down MS, a volume of 10
lL of the Glu-C crude
sample digestions was loaded onto a BioBasic-18 column (5
lm,
taining 850 lL of enzyme (1.1 lM final concentration), plus 66 lL
2.1 ꢁ 150 mm, ThermoFisher) and analyzed by LC-nanoESI-MS/
MS using the chromatographic system described above. Peptides
were separated in a 60 min gradient of 5–80% buffer B (A = 0.1%
of MG (0.41 mM final concentration) with or without amines (final
concentration of 0.41 mM for Car and 0.21 mM for Sp and Spe),
were incubated in buffer phosphate (pH 7.4–9.4) for 16 h at
FA in water, B = 0.1% FA in acetonitrile) at 100 lL/min flow rate.
37 °C in the absence of light. The final volume was 1500 lL. The
The Advion Triversa nanomate was set to collection mode allowing
simultaneous online nanoESI ionization and fraction collection.
Spray voltage and delivery pressure in the nanomate source were
set to 1.75 kV and 0.3 psi, respectively. The LTQ-FT ultra mass spec-
trometer was operated in positive polarity mode. Capillary voltage,
concentration of amino groups (Lys residues + amino terminal)
was 0.041 mM, so that the amino-group/MG/amine ratio was
1:10:10 (Car) or 1:10:5 (Sp, Spe). The activity of the enzyme was
assayed at 0 and 16 h.

P. Flores-Morales et al. / Bioorg. Med. Chem. 19 (2011) 1613–1622
1621
tube lens and capillary temperature were tuned to 35 V, 135 V and
200 °C, respectively. Acquisitions were performed in data-depen-
dent mode. Survey full-scan MS spectra (m/z 295-2000) were ac-
quired in the FT at 100,000 resolution (after accumulation of a
target value of 106). The three most intense ions were sequentially
isolated for fragmentation and detection in the linear ion trap
using collision induced dissociation (CID) at a target value of
50,000, 1 microscan averaging and a normalized collision energy
(CE) of 35%. Target ions already selected for MS/MS were dynami-
cally excluded for 30 s. Further CID analyses were also performed
off-line using the nanomate source by infusing the LC-collected
fractions of interest. A normalized CE of 35–42% was applied to
the ion trap isolated ions with high resolution (100,000) detection
in the FT and 200–1000 scan averaging.
atomic structure of the molecule, and the electrostatic potential
is determined by using the Poisson–Boltzmann equation.66 The
dielectric constant for the solvent was 80, and for the protein
was ranged from 4 to 20. Partial atomic charges were taken from
the AMBER (parm99)67 force field, and atomic radii from the opti-
mized values developed by Poisson–Boltzmann computations with
AMBER charges.68 The ionic strength was fixed at 0.1 M. The pro-
tein was initially located in the geometric center (101 Å3 box; grid
step of 1.0 Å), and then refined by focusing the grid on the active
site (101 Å3 box; grid step of 0.5 Å).
Acknowledgements
The authors thank Prof. Neil Kelleher and his team (Chemistry
Department at University of Illinois) for technical assistance and
advice. Financial support from FONDECYT through Project Nos.
1050965, 1090460, and 1100881 is gratefully acknowledged. P.
Flores-Morales thanks CONICYT for a post-doctoral fellowship.
4.6. MS data analysis
Mass spectrometers were powered by Xcalibur software 2.07.
Xcalibur Xtract algorithms and ProMass software version 2.8
(ThermoFisher Scientific) were used for charged state ion deconvo-
lution to zero charged species. MS/MS high resolution spectra of in-
tact proteins were deconvoluted with the in-house on-line
automation cRawler software (Prof. N.L. Kelleher’s property). Bio-
works 3.3.1 and Protein Calculator from Xcalibur software were
used to calculate theoretical molecular weights of intact and di-
gested proteins. Single protein and Sequence gazer options from
Prosight PC 2.0 (ThermoFisher Scientific)60 software were used to
localize modified residues from experimental high resolution MS/
MS data, after fragment ions Xtract deconvolution to zero charged
monoisotopic masses. MS raw data files from digested proteins
were processed with Proteome Discoverer vs. 1.1 (ThermoFisher
Scientific) against G6PD sequence.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.044.
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