Báo cáo Y học: Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli pptx

Contribution of Lys276 to the conformational flexibility of the active
site of glutamate decarboxylase from
Escherichia coli
Angela Tramonti
1
, Robert A. John
2
, Francesco Bossa
1
and Daniela De Biase
1
1
Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’ and Centro di Studio sulla Biologia Molecolare del CNR, Rome, Italy;
2
Cardiff School of Biosciences, Cardiff, UK
Glutamate decarboxylase is a pyridoxal 5¢-phosphate-
dependent enzyme responsible for the irreversible a-decar-
boxylation of glutamate to yield 4-aminobutyrate. In
Escherichia coli, as well as in other pathogenic and non-
pathogenic enteric bacteria, this enzyme is a structural
component of the glutamate-based acid resistance system
responsible for cell survival in extremely acidic conditions
(pH < 2.5). The contribution of the active-site lysine
residue (Lys276) to the catalytic mechanism of E. coli
glutamate decarboxylase has been determined. Mutation of
Lys276 into alanine or histidine causes alterations in the
conformational properties of the protein, which becomes less
flexible and more stable. The purified mutants contain very
little (K276A) or no (K276H) cofactor at all. However,
apoenzyme preparations can be reconstituted with a full
complement of coenzyme, which binds tightly but slowly.

The observed spectral changes suggest that the cofactor is
present at the active site in its hydrated form. Binding of
glutamate, as detected by external aldimine formation,
occurs at a very slow rate, 400-fold less than that of the
reaction between glutamate and pyridoxal 5¢-phosphate in
solution. Both Lys276 mutants are unable to decarboxylate
the substrate, thus preventing detailed investigation of the
role of this residue on the catalytic mechanism. Several lines
of evidence show that mutation of Lys276 makes the protein
less flexible and its active site less accessible to substrate and
cofactor.
Keywords: glutamate decarboxylase; pyridoxal 5¢-phos-
phate; active-site lysine; site-directed mutagenesis;
Escherichia coli.
In all pyridoxal 5¢ phosphate (PLP)-dependent enzymes
studied so far, the e-amino group of a conserved lysine
residue at the active site [1] binds the cofactor as a
Schiff’s base. It has been suggested that the formation of
an internal aldimine between the coenzyme and a
primordial apoenzyme occurred early in the evolution
of PLP-enzymes because, before becoming catalytically
advantageous, it prevented rapid loss of PLP which was
precious because of its ability to catalyse a number of
reactions on a wide variety of biosubstrates by itself [2].
Site-directed mutagenesis supports the proposal that
participation of this lysine residue in an internal aldimine
with the cofactor also accelerates formation of the
external aldimine between the PLP 4¢-aldehyde and
substrate amino groups because transaldimination is
more rapid than de novo Schiff’s base formation [3–6].

It also facilitates the proton transfers essential to many
B6-dependent reactions [3–10]. In the amino acid decar-
boxylases so far investigated, the corresponding lysine
appears not to be involved in reprotonation after
decarboxylation, but mainly to play a role in the initial
transaldimination, in proper positioning of the a-carb-
oxylate for decarboxylation and in product release
[11,12].
Bacterial glutamate decarboxylase (Gad, E.C. 4.1.1.15) is
one of the structural components of the glutamate-based
acid resistance system, responsible for acid survival of
enteric pathogens, such as Escherichia coli, Shigella flexneri
and Listeria monocytogenes [13–15], and of other nonpatho-
genic bacteria [16,17]. E. coli synthesizes two Gad isoforms,
GadA and GadB, 98% identical in amino acid sequence
and biochemically indistinguishable [18,19]. Gad catalyses
the irreversible a-decarboxylation of
L
-glutamate to yield 4-
aminobutyrate and CO
2
. It has been suggested that in this
enzyme the active-site lysine is involved in the protonation
of the quinonoid intermediate at C-4¢ during the abortive
decarboxylation–transamination reaction, while a histidine
has been proposed as the residue responsible for the
protonation at C-a which occurs during the physiological
decarboxylation reaction [20]. Site-directed mutagenesis
established that His167 and His275, likely candidates as
proton donors, are not responsible for the reprotonation

after CO
2
elimination [21].
The present work has been undertaken with the aim of
determining the contribution of the active-site lysine residue
(Lys276) to coenzyme binding and to stages in the reaction
catalysed by E. coli Gad.
Correspondence to D. De Biase, Dipartimento di Scienze Biochimiche
ÔA. Rossi FanelliÕ, Piazzale Aldo Moro, 5-00185 Roma, Italy.
Fax: + 39 06 49917566, Tel.: + 39 06 49917692,
E-mail:
Abbreviations: Gad, glutamate decarboxylase; PLP, pyridoxal
5¢-phosphate.
Enzymes: glutamate decarboxylase (P28302) (E.C.4.1.1.15).
Note: a web site is available at />homein.html
(Received 21 May 2002, revised 24 July 2002, accepted 26 July 2002)
Eur. J. Biochem. 269, 4913–4920 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03149.x
MATERIALS AND METHODS
Analytical reagents
Vent polymerase was from New England Biolabs. Restric-
tion enzymes, T4 DNA ligase and the agarose gel DNA
extraction kit were from Roche. The T7 sequencing kit,
DEAE-SepharoseÒ and Sephadex G-25Ò were from Phar-
macia. [a-
35
S]dATP (1000 CiÆmmol
)1
)wasfromNew
England Nuclear. Ingredients for bacterial growth were
from Difco. Oligonucleotides were from Genenco. Gua-

nidineÆHCl, 2,2,2-trifluoroethylamine, aminoacetonitrile
bisulfate and Gabase were from Sigma. Other chemicals
were from Merck.
Site-directed mutagenesis
Site-directed mutagenesis was performed by overlap exten-
sion polymerase chain reactions [22], following the proce-
dure described in Tramonti et al.[21].Mutagenicprimers
were 5¢-GGCCATGCATTCGGTCTG-3¢,fortheGadB-
K276A mutant, 5¢-GGCCATCACTTCGGTCTG-3¢,for
the GadB-K276H mutant, and their complementary
sequences. Fragments EcoRV/HindIII, generated by digest-
ing the amplicons from the second polymerase chain
reaction, were subcloned into pQgadB [19]. The newly
inserted fragments of plasmid pQgadBK276A and
pQgadBK276H were sequenced on both strands.
Purification of mutant forms of Gad
Expression and purification of mutant enzymes were as
described for wild-type enzyme [19]. The E. coli strain
JM109(pREP4), known to produce low levels of endo-
genous GadA/B was used as host [19]. Preparations of the
mutant enzymes were treated with NaBH
3
CN to inactivate
any wild-type enzyme present.
Calorimetric and spectroscopic analyses
Thermal unfolding of GadB-K276A and GadB-K276H
(1.5–2.0 mgÆmL
)1
) was analyzed under nitrogen pressure
on a MicroCal MC-2D differential scanning calorimeter

(MicroCal, Inc., Northampton, MA, USA). Results were
corrected for instrumental baseline and normalized for
protein concentration.
Absorption spectra were measured on a Hewlett-Packard
model 8452 diode-array spectrophotometer. CD spectra
were recorded as the average of three scans on a Jasco 710
spectropolarimeter equipped with a DP520 processor at
25 °C. Fluorescence spectroscopy was performed with a
LS50B fluorimeter (Perkin Elmer) at the excitation wave-
length of 295 nm. Curve fitting and statistical analysis were
carried out using the data manipulation software
SCIENTIST
(Micromath,SaltLakeCity,UT).ThePLPcontentofall
the enzyme preparations was determined by treating the
protein with 0.1
M
NaOH and measuring absorbance at
388 nm (e
388
¼ 6550 LÆmol
)1
Æcm
)1
[23]). The pH-depend-
ent absorbance variation of wild-type and mutant enzymes
was analyzed using the following equation:
Abs
H
n
E

À Abs
E
Abs À Abs
E
À 1 ¼
10
ÀnpK
10
ÀnpH
ð1Þ
where Abs
HnE
and Abs
E
are the absorbances of completely
protonated and unprotonated forms of enzyme, K is the
intrinsic dissociation constant and n is the number of
protons involved in titration.
The change in absorbance observed in the reaction of
GadB-K276A and GadB-K276H mutants with glutamate
was analyzed with Eqn (2) which describes two consecutive
irreversible reactions of the type A !
k
1
B !
k
2
C.
b ¼ a
0

Â
k
1
k
2
À k
1
Âðe
Àk
1
Ât
À e
Àk
2
Ât
Þð2Þ
In the above equation, b is concentration of the 412-nm
absorbing species (B) and a
0
is the concentration of species
Aattime0.
Gad activity assay
Enzyme activity was assayed by quantitating the reaction
product, 4-aminobutyrate, by HPLC [24] or using Gabase,
a commercial preparation containing 4-aminobutyrate
aminotransferase and succinic semialdehyde dehydrogen-
ase, as previously described [19]. On some occasions,
enzyme activity was assayed using the pH indicator
bromocresol green (0.02% w/v) as described by De Biase
et al. [25].

RESULTS
Physical properties of mutant enzymes
Yields of the mutant enzymes, GadB-K276A and GadB-
K276H, after the standard purification, i.e. in absence of
added PLP, were 50 mgÆL
)1
of bacterial culture, as for
wild-type GadB [19]. The mutant forms were stable for
several months at 4 °C. As judged by CD spectroscopy in
the far-UV region, mutations did not affect the overall
protein conformation. The transition temperature of
reconstituted GadB-K276A was 62.3 °C, suggesting that
this mutant enzyme adopts a significantly more stable
conformation than wild-type GadB (51 °C). The transi-
tion temperature of GadB-K276H (55.3 °C) was only
slightly higher than that of the wild-type enzyme (Fig. 1).
In support of the above observation, limited proteolysis
by trypsin showed that GadB-K276A is more resistant to
proteolytic degradation than the wild-type enzyme (data
not shown).
Spectral properties of GadB-K276A and -K276H
mutants
Absorption spectra of purified GadB-K276A showed
maxima at 280 and 328 nm (Fig. 2A). However, the specific
absorbance at 328 nm due to the cofactor was significantly
lower than that of the wild-type enzyme and, correspond-
ingly, the amount of PLP released by NaOH treatment was
only 10% of that expected for a fully saturated holoenzyme.
Nevertheless, the small amount of cofactor present was not
displaced by either gel filtration in 0.5

M
KH
2
PO
4
,or
incubation with cysteine or dialysis in 2
M
guanidineÆHCl.
Complete removal of the cofactor was achieved by
overnight dialysis against 1
M
KH
2
PO
4
,pH4.2,withthe
resulting apoenzyme having the spectrum shown in Fig. 2A,
inset. Purification of GadB-K276H following the standard
4914 A. Tramonti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
protocol yielded 100% apoenzyme. Unlike the wild-type
apoenzyme, which precipitates instantaneously upon coen-
zyme removal, both GadB-K276A and GadB-K276H
apoenzymes remained stable for many weeks in 0.1
M
sodium acetate, pH 4.6. For both mutant forms, the
holoenzyme was regenerated by treating the apoenzyme
(150 l
M
) overnight with a fivefold molar excess of PLP

(750 l
M
). Unbound cofactor was removed by extensive
dialysis against 0.1
M
sodium acetate, pH 4.6. The recon-
stituted mutant enzyme (Fig. 2A) contained one molecule
of PLP per monomer, as judged by NaOH treatment. The
absorption spectrum above 320 nm fitted well to the sum of
two log normal curves having k
max
values of 330 nm and
388 nm (Fig. 2B). The great majority of the coenzyme was
present as 330 nm-absorbing chromophore, the corres-
ponding peak being broader, but less intense, in the GadB-
K276H mutant enzyme (Fig. 2B). Continuous monitoring
of the absorbance changes associated with reconstitution of
GadB-K276A indicated that the absorbance decrease at
388 nm (free PLP) and the increase at 330 nm were biphasic
(data not shown) and the curve fitted well to the sum of
two exponentials (k
1
¼ 0.58 ± 0.001 min
)1
; k
2
¼ 0.059 ±
0.002 min
)1
) with the more rapid phase accounting for 70%

of the reaction.
Treatment of both reconstituted mutants with
NaCNBH
3
did not affect the spectra, demonstrating that
PLP is bound as the free aldehyde.
Figure 2(C) shows CD spectra of wild-type and GadB
mutants in the 300–500 nm range, where the chromophore
of all enzymic forms absorbs maximally. Notably, the
GadB-K276A mutant produced a much smaller CD signal
than the GadB-K276H mutant, despite the lower absorb-
ance of the latter (Fig. 2B).
E. coli Gad undergoes well-established changes in activity
and in the absorption spectrum of the cofactor depending
on pH (Fig. 3A) [26]. At pH values higher than 5.3, the
enzyme absorbs maximally at 340 nm and is inactive,
whereas at lower pH values, the enzyme absorbs maximally
at 420 nm. The change in activity parallels the absorbance
Fig. 2. Absorption and CD spectra of GadB Lys276 mutants. (A)
Absorption spectra of GadB-K276A mutant. The absorption spectra
of GadB-K276A (20 l
M
) as it is purified under standard conditions
(dotted line), in the apoenzymatic form (solid line) and after its
reconstitution with PLP (dashed line) were determined in 0.1
M
sodium
acetate, pH 4.6, containing 0.1 m
M
dithiothreitol. (B) Analysis of

absorption spectra of GadB-K276A and GadB-K276H mutants. The
solid lines are those of best fit to the sum of two log normal curves [32]
having k
max
values of 330 nm and 388 nm. Only one in three of the
data points collect for the GadB-K276A (d) and GadB-K276H (j)
absorption spectra is shown. (C) CD spectra of wild-type and mutant
enzymes. The CD spectra of wild-type GadB (solid line), and of GadB-
K276A (dotted line) and GadB-K276H (dashed line) mutants, each at
a concentration of 184 l
M
, were determined in 50 m
M
sodium acetate,
pH 4.6, containing 0.1 m
M
dithiothreitol.
Fig. 1. Differential scanning calorimetry of wild-type GadB and active-
site lysine mutants. Thermal denaturation profiles of GadB wild-type
(solid line), of GadB-K276A mutant (dashed line) and GadB-K276H
mutant (dotted line). Protein samples (1.5–2.0 mgÆmL
)1
)werein0.1
M
sodium acetate, pH 3.6, containing 0.1 m
M
dithiothreitol.
Ó FEBS 2002 Role of Lys276 in E. coli glutamate decarboxylase (Eur. J. Biochem. 269) 4915
change and it is clear from the pH profile that multiple
protons are involved in the transition (Fig. 3A, inset) [26]. In

both active-site lysine mutants, changing the pH from 3.6 to
6.4 induced an increase of the species absorbing at 388 nm
and a decrease of that absorbing at 328 nm (Fig. 3B). The
systematic changes in the spectrum of the mutant enzymes
showed the proportion of the 388 nm chromophore
increasing towards a limiting value. Again, as in the wild-
type enzyme (Fig. 3A), the abrupt changes in absorbance
with increasing pH did not fit well to a single ionization
event and the data from both active site-lysine mutants
fitted adequately to a model that required the simultaneous
loss of 4–6 protons (Fig. 3B, inset).
Experiments in absorption and fluorescence spectroscopy,
in the presence of increasing concentrations of guani-
dineÆHCl (0–6
M
), were conducted with both wild-type and
the mutant enzymes. In both mutants, guanidineÆHCl in the
range 0–2
M
induced an absorbance change characterized
by an increase at 388 nm and a decrease at 328 nm
(Fig. 3C). The same behavior was also observed when
sodium chloride was added (data not shown). In the same
concentration range of guanidineÆHCl and sodium chloride,
the absorbance spectrum of the wild-type enzyme remained
unaffected.
When excited at 295 nm in the absence of guanidineÆHCl,
the reconstituted mutant enzymes exhibited two fluores-
cence emission maxima (Fig. 4A). The first, at 332 nm, also
present in the wild-type (Fig. 4B), is due to the intrinsic

fluorescence of the protein. It is likely that the second, at
380 nm, is due to energy transfer to the 330-nm absorbing
form of PLP at the active site. At 2
M
guanidineÆHCl the
emission spectrum of both mutants showed exclusively a
peak at 332 nm, because the PLP in the active site has been
converted into a form mainly absorbing at 388 nm. In the
range 2–5
M
guanidineÆHCl the change in fluorescence
emission spectra indicated that the unfolding profiles of
wild-type and mutant enzymes are superimposable, with the
transition point (50% unfolding) centered at 3.4
M
guani-
dineÆHCl. Upon unfolding, a blue-shifted emission maxi-
mum at 360 nm in both wild-type and mutant enzymes was
observed (Fig. 4).
Reaction with glutamate
Addition of 20 m
M
glutamate to GadB-K276A produced
an increase in absorbance at 412 nm and a decrease at
328 nm each with the same half-time of approximately 2 h
(Fig. 5A). The change was characterized by an isosbestic
point at 342 nm. The 412 nm contribution was completely
abolished by adding NaCNBH
3
, a reagent known to reduce

exclusively the protonated Schiff bases. After 7 h, an
additional slow spectral change occurred which was com-
plete within 30 h. This spectral change is characterized by a
decrease at 412 nm and an increase at 340 nm, with an
isosbestic point at 375 nm (Fig. 5B). The change observed
at 412 nm conformed to an equation describing two
consecutive irreversible reactions (see Materials and meth-
ods, Eqn 2). Increasing the glutamate concentration
produced a linear increase in the value observed for k
1
whereas k
2
did not change significantly (0.07 ± 0.01 h
)1
).
No 4-aminobutyrate was detected at the end of the reaction
although the method used was sensitive enough to detect
this compound at 10% of the enzyme concentration.
Fig. 3. Effect of pH and guanidineÆHCl on the absorbance spectra. (A)
Absorption spectra of wild-type GadB (11.4 l
M
) were determined in
0.1
M
sodium acetate in the pH range 3.5–6.2. Only relevant spectra are
shown. In the inset, the pH variation at 420 nm is represented. The
solid line is that of best fit to Eqn (1) (Materials and methods), with
pK ¼ 5.292 ± 0.007, Abs
E
¼ 0.0069 ± 0.0008, Abs

HnE
¼ 0.1058 ±
0.0007 and n ¼ 5.1 ± 0.4. (B) Absorption spectra of GadB-K276H
(10.3 l
M
) were determined as above. In the inset, the pH variation at
388nmisrepresented.ThesolidlineisthatofbestfittoEqn(1)
(Materials and methods), with pK ¼ 5.60 ± 0.01, Abs
E
¼ 0.032 ±
0.001, Abs
HnE
¼ 0.0060 ± 0.0006 and n ¼ 8.4 ± 4.6. (C) Absorption
spectra of GadB-K276H mutant (19 l
M
) measured in the presence of
0, 0.1, 0.2, 0.4, 0.6, 1 and 2
M
guanidineÆHCl in 0.1
M
sodium acetate,
pH 4.6. The pH- and guanidine-dependent absorbance changes in the
GadB-K276A mutant were identical with those in GadB-K276H
mutant, and therefore the data are omitted.
4916 A. Tramonti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Moreover, no pH increase was detected during the reaction
with glutamate when using the pH indicator bromocresol
green in an unbuffered solution, thus indicating that there
was no consumption of protons. Treatment of the reaction
mixture with 0.2

M
NaOH at the times 0, 5 and 20 h
released the full complement of cofactor as PLP (detected
and measured by its 388 nm absorbance).
Reaction of GadB-K276H with 20 m
M
glutamate resulted
in a spectral change similar to that occurring in the GadB-
K276A mutant (data not shown). The only difference
observed between the two mutants was the amplitude of
the change in absorbance at 412 nm, which at 20 l
M
protein
was much smaller in GadB-K276H (total absorbance change
of 0.03) than in GadB-K276A (total absorbance change of
0.12; Fig. 5A). As observed for the alanine mutant, the
histidine mutant did not produce 4-aminobutyrate.
In the active-site lysine mutants of aspartate aminotrans-
ferase [7], tryptophan synthase [4],
D
-amino acid transam-
inase [5] and alanine racemase [27] it was observed that
exogenous amines can partially or totally substitute for
the catalytic role of the active-site lysine. In order to
study the effect of exogenous amines on GadB-K276A, 2,2,
2-trifluoroethylamine (pK
a
¼ 5.7) [7] and aminoacetonitrile
bisulfate (pK
a

¼ 5.3) [7] were added to the enzyme in
presence of sodium glutamate.
When 1
M
2,2,2-trifluoroethylamine or 0.2
M
amino-
acetonitrile bisulfate were included in the reaction mixture
containing 20 l
M
GadB-K276A and 20 m
M
sodium
glutamate, the enzyme underwent spectral changes identical
to those already described, but the increase in absorbance at
412 nm occurred approximately six times faster. The devel-
opment of turbidity however, prevented analysis of the later
phases of the reaction. Even in the presence of exogenous
amines 4-aminobutyrate production was undetectable (data
not shown).
When both mutant enzymes were incubated with
glutamate in the presence of a low concentration of
guanidineÆHCl (0.4
M
), spectral changes identical to those
previously described occurred, even though the initial
spectrum was different, and at the end of reaction a
species absorbing at 340 nm could be detected (data not
shown).
DISCUSSION

Many of the alterations produced by mutating the active-
site Lys276 of GadB can be attributed to changes in the
conformational properties of the protein. The findings that
the mutation to alanine increases the unfolding temperature
Fig. 5. Reaction of GadB-K276A with glutamate. The absorbance
spectra of GadB-K276A mutant (60 l
M
) were recorded with 20 m
M
sodium glutamate in 0.1
M
sodium acetate, pH 4.6, from 0 to 6 h (A)
and from 6 to 30 h (B).
Fig. 4. Effect of guanidine on fluorescence emission spectra. (A) Emis-
sion spectra (k
exc
¼ 295 nm) of GadB-K276H mutant (0.82 l
M
)in
0.1
M
sodium acetate, pH 4.6, containing 0, 1, 2, 3 and 5
M
guani-
dineÆHCl. (B) Emission spectra of wild-type GadB (0.77 l
M
)in0.1
M
sodium acetate, pH 4.6, containing 0, 2, 4 and 5
M

guanidineÆHCl.
Ó FEBS 2002 Role of Lys276 in E. coli glutamate decarboxylase (Eur. J. Biochem. 269) 4917
of the holoenzyme by 11 °C, makes it resistant to tryptic
hydrolysis and prevents the precipitation observed with the
wild-type apoenzyme demonstrates that the absence of this
lysine residue, either as an aldimine with the cofactor or as a
protonated primary amine in the apoprotein, makes the
protein less flexible. The same reduced flexibility seems
likely to account for the slow, but ultimately tight, binding
of PLP to the apoenzyme in vitro and for the fact that
preparations of the mutant enzymes are always largely as
apoenzyme. Similarly, inflexibility of the protein would also
explain the observation that PLP, bound to the mutant
enzymes, forms an aldimine with glutamate much more
slowly than free PLP.
The observation that the mutant apoenzymes are able to
form stable holoenzymes despite the absence of the active
site lysine residue shows that, as in other decarboxylases
[11,12], non–covalent interactions between protein and
cofactor are sufficient to ensure tight binding. This is also
in line with the finding that His275 contributes to cofactor
binding via an ionic interaction with the phosphate group of
PLP [21].
Because the mutant forms of GadB cannot form an
internal aldimine, it is not surprising that the 420 nm
chromophore, characteristic of the wild-type enzyme at
pH 4.6, is absent. However, the spectrum of PLP bound to
the Lys276 mutants is quite different from the spectrum of
the same compound when it is free in solution. Absorption
bands at 388 nm and 330 nm are present in the spectra of

both free PLP and PLP bound to the mutant enzymes, but in
PLP free in solution the 388 nm chromophore is the most
abundant species, whereas it is only a minor component of
the spectrum of the mutant enzymes. In free PLP, the
388 nm and 330 nm chromophores are attributed to the
unsubstituted and hydrated aldehydes, respectively [28]. It
seems likely that the absorbance changes observed are due to
an increase in the proportion of the PLP hydrate when the
cofactor binds. A similar structure has also been suggested to
be formed in the active-site lysine mutant of aromatic
L
-amino acid decarboxylase [11]. The biphasic nature of
the changes in spectrum occurring upon PLP binding,
reported also with the wild-type enzyme [29], suggests that
initial cofactor binding is followed by a slower confor-
mational adjustment. The CD spectra (Fig. 2C) show
that GadB-K276H retains much more asymmetry than
GadB-K276A probably because movement of the cofac-
tor within the active site is more restricted by the histidine
side-chain.
Both wild-type GadB and its active-site lysine mutants
show abrupt pH-dependent spectral changes involving
simultaneous transfer of multiple protons. Other PLP-
dependent enzymes undergo similar pH-dependent changes
which are related to activity and are attributed to protona-
tion of the internal aldimine formed with lysine and the
cofactor aldehyde. For example, aspartate aminotransferase
is converted from an inactive 430 nm-absorbing protonated
internal aldimine to an active unprotonated 362 nm-
absorbing form with a pK of 6.2 in a process that fits well

to the ionization of a single proton [30]. It has been
suggested that in wild-type Gad, the ionization responsible
for the absorbance change does not take place on the
internal aldimine and much evidence indicates that the
change in spectrum of the wild-type enzyme is due to a
conformational transition in the protein induced by shifting
the pH [31]. It seems very likely that the pH-dependent
changes that occur in the spectrum of the mutants under
investigation in the present work are due to the same pH-
induced conformational transition observed in the wild-type
and that the different forms of the cofactor present in wild-
type and mutant enzymes are recording the same event at
the active site. The observation that the pH-dependent
spectral changes occur in enzyme forms without the internal
aldimine demonstrates that the protonation responsible for
the absorbance changes and for activation of the wild-type
enzyme is not of the internal aldimine itself.
To explain the pH-dependent occurrence of the
330 nm-absorbing chromophore, it has been proposed
that, in the wild-type enzyme, high pH induces the
formation of an aldamine between the internal aldimine
and an enzyme cysteine residue and that the low pH
conformation favors the unsubstituted internal aldimine
[31]. However, because in the mutant enzymes the high
pH favors the 388 nm-absorbing unsubstituted aldehyde,
formation of a covalent bond between PLP and a cysteine
side-chain can be excluded as the basis of the pH-
dependent absorbance changes observed with the Lys276
mutant enzymes. An explanation that unites observations
from both wild-type and mutant enzymes is that the pH-

dependent conformational change induces an alteration in
the polarity of the active site. In this hypothesis, the
environment of the cofactor is more hydrated in the low-
pH conformation. Thus, the increased polarity favors the
420 nm-ketoenamine tautomer in the wild-type enzyme
and the 330 nm-absorbing hydrated form of PLP in the
mutant enzymes. Conversely, the less hydrated environ-
ment of the high-pH conformation favors the 340 nm-
absorbing enolimine tautomer of the wild-type cofactor
and the 388 nm-absorbing unhydrated aldehyde of PLP in
the mutant enzymes (Fig. 6)
1
.
Fig. 6. Chemical structures of the chromophore proposed to be present
at the active site of GadB wild-type and GadB-K276 mutants at low and
high pH, respectively.
4918 A. Tramonti et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In the presence of low concentrations of guanidineÆHCl
or of sodium chloride, there is a spectral change in the
mutant enzymes similar to that which occurs upon changing
the pH. We suggest that low concentrations of solutes, by
subtracting water molecules, cause a change in polarity of
the active site and favor the 388 nm-absorbing unhydrated
aldehyde of PLP.
The failure of all the methods used to detect enzymatic
activity in GadB-K276A and GadB-K276H mutants
shows that GadB mutated at the active-site lysine loses
reactivity towards the substrate, though the mutants are
still capable of slowly binding
L

-glutamate and forming an
external aldimine. The increase in absorbance at 412 nm
observed when GadB K276A was mixed with glutamate,
together with the observation that this chromophore
converted to one absorbing at 340 nm when NaCNBH
3
was added provides strong evidence that an external
aldimine is formed between the cofactor and the amino
acid. The linear dependence on glutamate concentration of
the observed rate constant governing this phase shows
that there is no detectable saturation of the mutant
enzyme with substrate, even at high concentrations. The
second order constant calculated from this experiment
(3.6 · 10
)3
±0.3 · 10
)3
M
)1
Æs
)1
) is much lower than
that calculated for the reaction between free PLP and
glutamate in 0.1
M
sodium acetate, pH 4.6 (1.47 ±
0.13
M
)1
Æs

)1
). The 400-fold reduction in reaction rate
contrasts markedly with the observation that PLP bound
to the corresponding mutant of aspartate aminotransfer-
ase forms an external aldimine with glutamate or aspartate
at least three orders of magnitude more rapidly than does
free PLP [3]. It seems likely that at least part of this major
reduction in reactivity of the enzyme-bound PLP is due to
the extensive hydration we propose to be responsible for
the predominant 330 nm chromophore, as well as to the
reduced flexibility discussed earlier.
The cause of the subsequent and even slower change in
spectrum from 412 nm to 340 nm is unknown, although
the failure to detect 4-aminobutyrate shows that it is not
due to decarboxylation of the substrate. A possible
explanation could be that Lys276 is, either directly or
indirectly, involved in correctly positioning the Ca-COO
–
bond orthogonal to the plane of the delocalized cofactor
imine system. Moreover, the release of cofactor as PLP
when the enzyme was treated with NaOH after comple-
tion of this reaction shows that it is not due to
transamination to pyridoxamine phosphate. A similar
but much more rapid spectral change occurs in the
reaction catalyzed by the wild-type enzyme, where it has
been attributed to a reversible conformational change in
the enzyme leading to a form that does not undergo
further reaction [19,21].
ACKNOWLEDGMENTS
This work was partially supported by grants from the Italian Ministero

dell’Istruzione, dell’Universita
`
e della Ricerca and from the Istituto
Pasteur-Fondazione Cenci Bolognetti (to DDB). The Centro di
Eccellenza di Biologia e Medicina Molecolare (BEMM), Universita
`
di Roma La Sapienza, is also acknowledged.
We thank Professor A. Giartosio for DSC measurements and
Professor D. Barra for critical reading of the manuscript.
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