Báo cáo Y học: Pyruvate decarboxylase from Kluyveromyces lactis An enzyme with an extraordinary substrate activation behaviour pptx

Pyruvate decarboxylase from
Kluyveromyces lactis
An enzyme with an extraordinary substrate activation behaviour
Florian Krieger*, Michael Spinka, Ralph Golbik, Gerhard Hu¨ bner and Stephan Ko¨ nig
Institut fu
¨
r Biochemie, Fachbereich Biochemie/Biotechnologie, Martin-Luther-Universita
¨
t Halle-Wittenberg, Halle/Saale, Germany
Pyruvate decarboxylase (EC 4.1.1.1) was isolated and puri-
fied from the yeast Kluyveromyces lactis. The properties of
this enzyme relating to the native oligomeric state, the sub-
unit size, the nucleotide sequence of the coding gene(s), the
catalytic activity, and protein fluorescence as well as circular
dichroism are very similar to those of the well characterized
pyruvate decarboxylase species from yeast. Remarkable
differences were found in the substrate activation behaviour
of the two pyruvate decarboxylases using three independent
methods: steady-state kinetics, stopped-flow measurements,
and kinetic dilution experiments. The dependence of the
observed activation rate constant on the substrate concen-
tration of pyruvate decarboxylase from K. lactis showed a
minimum at a pyruvate concentration of 1.5 m
M
. According
to the mechanism of substrate activation suggested this local
minimum occurs due to the big ratio of the dissociation
constants for the binding of the first (regulatory) and the
second (catalytic) substrate molecule. The microscopic rate
constants of the substrate activation could be determined by
a refined fit procedure. The influence of the artificial acti-

vator pyruvamide on the activation of the enzyme was
studied.
Keywords: kinetics; thiamine diphosphate; microscopic rate
constants; pyruvamide
The cytosolic pyruvate decarboxylase (PDC) is a key
enzyme at the branching point of alcoholic fermentation
and respiration in yeast, some bacteria and plant seeds.
It catalyses the nonoxidative decarboxylation of pyruvate
to acetaldehyde and carbon dioxide. Thiamine diphos-
phate (TDP) and Mg
2+
are both required as cofactors in
this reaction. In yeast and bacteria, the catalytically
active enzyme is composed of four subunits. PDC species
from plant seeds are able to form higher oligomeric
states [1–3]. In the genome of the yeast Kluyveromyces
lactis one gene was found to code for PDC [4]. Its
nucleotide sequence has 85% identity to PDC1 from
Saccharomyces cerevisiae. All amino acids that are likely
to be involved in the regulation and catalysis of PDC are
conserved. The phenomenon of substrate activation has
been described for all PDC species investigated so far,
except for the enzyme from Zymomonas mobilis. In 1978,
Hu
¨
bner et al. [5] described the kinetics of substrate
activation of ByPDC (PDC from brewer’s yeast) showing
a hyperbolic dependence of the activation rate constant
on the substrate concentration. Furthermore, it was
demonstrated that the artificial substrate surrogate

pyruvamide is able to activate PDC. The following
minimal model of the catalytic mechanism was derived
[6]:
Scheme 1.
A substrate molecule binds rapidly to a regulatory
site of the inactive enzyme E
i
and triggers an isomeri-
zation towards an active enzyme conformation SE
a
.In
a subsequent step the active conformation state binds a
second substrate molecule and catalyses its decarboxy-
lation to yield acetaldehyde (AA). The isomerization
step proceeds slowly compared to the substrate binding.
K
a
is the dissociation constant of the substrate binding
to the regulatory site preceding the isomerization.
The isomerization constant K
iso
is equal to the ratio
k
–iso
/k
iso
.TheK
m
value for substrate conversion is
defined as:

K
m
¼
k
3
Áðk
2
þ k
À1
Þ
k
1
Áðk
2
þ k
3
Þ
Correspondence to S. Ko
¨
nig, Institut fu
¨
r Biochemie, Fachbereich
Biochemie/Biotechnologie, Martin-Luther-Universita
¨
t
Halle-Wittenberg, Kurt-Mothes-Str. 3, 06099 Halle/Saale, Germany.
Fax: + 345/5527014, Tel.: + 345/5524829,
E-mail:
Abbreviations: PDC, pyruvate decarboxylase (2-oxo acid carboxy
lyase, EC 4.1.1.1); ByPDC, pyruvate decarboxylase from brewer’s

yeast; KlPDC, pyruvate decarboxylase from Kluyveromyces lactis;
PsPDC, pyruvate decarboxylase from Pisum sativum;ScPDC,
pyruvate decarboxylase from Saccharomyces cerevisiae;ZmPDC,
pyruvate decarboxylase from Zymomonas mobilis;TDP,thiamine
diphosphate.
*Present address: Biozentrum, Universita
¨
t Basel, Departement
Biophysikalische Chemie, Klingelbergstrasse 70, 4056 Basel,
Switzerland.
Note: a web site is available at />(Received 20 March 2002, accepted 17 May 2002)
Eur. J. Biochem. 269, 3256–3263 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03006.x
A comparison of the crystal structures of native and
pyruvamide-activated ByPDC clearly demonstrated that
this isomerization is realized by a rearrangement of the
dimers within the tetramer. This 30° rotation resulted
in a disorder-order transition of two loop regions and
thus in closing two of four active sites of the enzyme
[7,8].
Here, PDC from K. lactis was characterized. The
substrate activation behaviour of this enzyme displayed a
complex dependence of the activation rate constant on the
substrate concentration. The dissociation constants of the
substrate at the regulatory and the catalytic site were
determined and compared to other PDC species.
MATERIALS AND METHODS
Chemicals
All reagents used for enzyme purification and activity
measurements were of analytical grade and purchased from
Merck, Serva, and Sigma–Aldrich. Columns and media

were from Amersham Pharmacia Biotech.
Yeast strain and media
The K. lactis strain JA-6 was a gift from I. Eberhardt
(Laboratorium of Molecular Cell Biology, Biological
Department, Katholic University Leuven, Belgium; present
address, Department of Molecular Biology, Gent Univer-
sity, Belgium). The yeast complete medium contained 1%
(w/v) yeast extract, 2% (w/v) peptone, 5% (w/v) glucose,
0.1 m
M
thiamine and 0.1 m
M
magnesium sulfate. The yeast
cultures (1 L) were inoculated with precultures (10% of the
volume)andgrewforatleast24hat29°Catashaker
frequency of 110 r.p.m.
Purification of PDC from
K. lactis
The purification procedure was developed on the basis of
methods established for other PDC from other species [2,9–
11]. All steps were carried out at 4 °C. Frozen K. lactis cells
(60 g) were thawed in 200 mL of 100 m
M
sodium phos-
phate pH 6.1, 5 m
M
dithiothreitol, 0.1 m
M
TDP, 0.1 m
M

magnesium sulfate, 5% (v/v) glycerol, 10 l
M
phenyl-
methylsulfonyl fluoride and disrupted using glass beads
(0.3–0.5 mm diameter) in a beat beater homogeniser (6
times for 30 s separated by 5 min cooling periods). The glass
beads were washed three times with 50 mL buffer. After
centrifugation (14 500 g for 30 min), 0.75% (w/v) strepto-
mycin sulfate was added to the supernatant under continu-
ous stirring at 4 °C for 30 min. The solution was centrifuged
at 14 500 g for 30 min; the precipitate was discarded and
27% (w/v) ammonium sulfate was added to the superna-
tant. After centrifugation at 4 °C, 10% (w/v) ammonium
sulfate was added to the supernatant. The solution was
stirred for 30 min and centrifuged again. The pellet was
resuspended in 20 mL of 100 m
M
Mes/NaOH pH 6.0,
150 m
M
ammonium sulfate. Insoluble protein was removed
by centrifugation at 20 000 g for 15 min. The supernatant
was loaded on a Sephacryl S200 H column (5.0 · 100 cm,
flow rate 1 mLÆmin
)1
) equilibrated and eluted with the same
buffer. Fractions containing KlPDC (PDC from K. lactis)
activity were precipitated by 50% (w/v) ammonium sulfate.
The pellet was resuspended in 10 mL of 20 m
M

Bistris
pH 6.8. The solution was desalted on a Superdex G50
column (1.6 · 30 cm, flow rate 1 mLÆmin
)1
) and applied to
an anion exchange column Resource Q (6 mL, flow rate
1mLÆmin
)1
). The protein was eluted using an increasing
ammonium sulfate gradient (0–0.2
M
, 100 mL). Fractions
containing KlPDC were stabilized by 1
M
ammonium
sulfate and loaded on a hydrophobic interaction column
Resource Phe (1 mL, flow rate 1 mLÆmin
)1
.). The protein
was eluted using a stepwise decreasing ammonium sulfate
gradient. KlPDC was detected at ammonium sulfate
concentrations below 400 m
M
. The fractions containing
highly purified KlPDC were precipitated by 50% (w/v)
ammonium sulfate and stored at )20 °C.
Enzyme assay and protein determination
Catalytic activity was measured in 0.1
M
Mes/NaOH

pH 6.0, 5 m
M
TDP, 5 m
M
magnesium sulfate at 340 nm
and 30 °C (Uvikon 941, Kontron Instruments) using the
established coupled optical test [12] with alcohol dehydro-
genase from yeast (alcohol dehydrogenase, Sigma,
45 UÆmL
)1
) and NADH at enzyme concentrations of
5–12.5 lgÆmL
)1
. At a substrate concentration above 40 m
M
the catalytic activity was measured at 366 nm. In order to
ensure that the lag phase in product formation was not due to
insufficient activity of the auxiliary enzyme alcohol dehy-
drogenase, its concentration was varied between 4 and
45 UÆmL
)1
; no effect on the duration of the lag phase was
observed. The PDC activity in the reaction mixture was not
larger than 0.4 UÆmL
)1
and the activity of alcohol dehy-
drogenase was determined for the reverse reaction that is
about 20 times higher in the direction from the aldehyde to
the alcohol. Consequently, the auxiliary enzyme should not
limit the substrate activation of KlPDC.

Substrate activation measurements were performed on
a stopped-flow spectrophotometer (SX.18MV, Applied
Photophysics) using the same buffer mentioned above.
One syringe contained the substrate pyruvate and the
auxiliary enzyme alcohol dehydrogenase (Sigma,
1mgÆmL
)1
, respectively, 900 UÆmL
)1
) and NADH, the
other KlPDC (50–100 lgÆmL
)1
), 10 m
M
TDP, and 10 m
M
magnesium sulfate. The solutions were mixed in a ratio of
1:1.
All dilution experiments were carried out in the same
buffer system mentioned above and started at a substrate
concentration of 2 m
M
. After 65 s the reaction mixture was
diluted manually by adding buffer containing the cofactors
(0.1
M
Mes/NaOH,pH6.0,5m
M
TDP, 5 m
M

magnesium
sulfate, 45 UÆmL
)1
alcohol dehydrogenase and 0.3–1.0 m
M
NADH)inaratioof1:2,1:3and1:5.Theenzyme
concentration was between 22.5 and 30 lgÆmL
)1
after
dilution.
The protein concentration in the crude extract was
determined according to Bradford [13] with bovine serum
albumin as standard protein. In all other cases the protein
concentration was calculated from the UV spectra at
280 nm using the molar extinction coefficient of
61 950
M
)1
Æcm
)1
for the KlPDC subunit, derived from
the amino-acid sequence using the software package of
EXPASY
.
Ó FEBS 2002 Substrate activation of K. lactis pyruvate decarboxylase (Eur. J. Biochem. 269) 3257
SDS/PAGE
SDS/PAGE (10% acrylamide) was carried out according to
the method of Laemmli [14]. Gels were stained with
Coomassie Brillant Blue G 250.
Determination of the molecular mass

This was performed on a Fractogel EMD BioSEC(S)
column (1.6 · 60 cm, Merck kgaA, flow rate 1 mLÆmin
)1
)
in 0.1 m
M
Mes/NaOH pH 6.0.
Mass spectrometry
Homogeneous KlPDC was desalted on a HiTrap column
(5 mL) using 10 m
M
ammonium acetate pH 6.4. Enzyme
solution (0.7 mgÆmL
)1
) was mixed with an equal volume
of 90 m
M
3,5-dimethoxy 4-hydroxycinnamic acid. The
molecular mass was detected on a REFLEX (Bruker-
Franzen Analytik) time of flight (TOF) mass spectrometer
with matrix-assisted laser desorption ionization (MALDI)
using a nitrogen laser at 337 nm and an acceleration voltage
of 30 kV. Bovine serum albumin (Merck) served as a
protein standard.
Theoretical background of substrate activation
For the studies on the mechanism of substrate activation Eqn
(1) can be applied to analyse the corresponding progress
curves. A
0
is the initial absorbance at zero time, D

SS
,the
steady-state velocity of absorbance change, D
0
, the corres-
ponding initial velocity at zero time, and k
obs
, the observed
first-order rate constant of the substrate activation.
A ¼ A
0
À D
SS
Á t þ
D
SS
À D
0
k
obs
Á½1 À expðÀk
obs
Á tÞ ð1Þ
The zero time slopes were found to be very small in the
absence of the activator pyruvamide (D
0
/D
SS
¼ 0.018).
Inclusion of D

0
, however, in Eqn (1) enables the application
of this equation to progress curves recorded in the presence
of pyruvamide. The dependence of the observed rate
constant on the substrate concentration given in Eqn (2)
[15] could be derived from Scheme 1.
k
obs
¼
k
Àiso
Á K
m
½SþK
m
þ
k
iso
Á½S
½SþK
a
ð2Þ
The dissociation constants are defined in Scheme 1. The
observed rate constant of substrate activation k
obs
is
composed of two functions. The first one, including K
m
,
describes a property of the catalytic centre of the enzyme,

the second, hyperbolic one, including K
a
, a property of the
regulatory substrate binding site. From Eqn (2), extrema of
k
obs
can be expected at a substrate concentration of
½S
ext
¼
K
m
Á
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
K
a
=K
iso
Á K
m
p
À K
a
1 À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
K
a
=K
iso
Á K

m
p
ð3Þ
In the case of K
a
/K
m
<K
iso
<K
m
/K
a
, a maximum will occur
and in the case of K
m
/K
a
<K
iso
<K
a
/K
m
, a minimum will
occur, otherwise [S]
ext
will be negative.
From Scheme 1 the following steady-state rate equation
can be derived:

vð½SÞ ¼
V
max
Á½S
2
K
a
Á K
iso
Á K
m
þ½SÁK
m
Áð1 þ K
iso
Þþ½S
2
¼
V
max
Á½S
2
A þ B Á½Sþ½S
2
ð4Þ
where: V
max
is the maximum velocity, A ¼ K
a
Æ K

iso
Æ K
m
,
the overall dissociation constant of the complex SE
a
S;
K
a
Æ K
iso
is the overall dissociation constant of the complex
SE
a
,andB¼ K
m
Æ (K
iso
+ 1). This function follows a
sigmoid shape characterized by an apparent Hill coefficient
between one and two. In the case of ÖA ) B, the Hill
coefficient is almost two, in the case of ÖA ( B, the Hill
coefficient is close to one. Therefore, the larger the ratio A/B,
the more pronounced the sigmoidicity of the dependence of
thesteady-staterateonthesubstrateconcentrationappears.
In terms of the activation process, this means that the
weaker the binding of the activating substrate molecule to
the regulatory centre and/or the more unfavoured is the
activated state against the inactive one, the larger the
deviation from the Michaelis–Menten behaviour.

From Eqn (4) the value S
0.5
, defining the substrate
concentration at which the velocity is V
max
/2, can be
obtained as
S
0:5
¼
B
2
þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
B
2
4
þ A
s
ð5Þ
The values k
)iso
and K
m
are calculated using Eqns (6) and
(7) deduced from Eqns (4) and (5):
k
Àiso
¼
k

iso
Á A
K
a
Á B À A
ð6Þ
K
m
¼
ðS
0:5
Þ
2
K
a
Á K
iso
þ S
0:5
Áð1 þ K
iso
Þ
ð7Þ
These equations are valid only for enzymes without
significant initial activity (D
0
/D
SS
close to zero). This is not
thecaseforPsPDC(D

0
/D
SS
¼ 0.25, from reference [16]).
However, we have involved this enzyme species in our
analysis for sake of comparison.
RESULTS AND DISCUSSION
Purification of PDC from
K. lactis
In contrast to brewer’s yeast, K. lactis is a Crabtree-
negative yeast exhibiting repressed alcoholic fermentation
under oxygen saturation. According to Breunig et al.[17],
K. lactis expresses PDC at high glucose and low oxygen
concentration. Considerably high amounts of KlPDC
were obtained under these growth conditions. A summary
of the purification procedure is illustrated in Table 1.
Twenty-five micrograms of purified PDC with a catalytic
activity of about 40 UÆmg
)1
were obtained from 60 g
yeast cells from 4 L of cell culture. The yield and catalytic
activity are comparable to those of PDC from other
organisms [1–3,9,10,18–22]. Only one type of subunit was
detectable in the SDS/PAGE (Fig. 1) in contrast to
ByPDC [9,23] and PDC species from plant seeds that
exhibit two types of subunits with slightly different sizes.
The molecular mass of 61.5 ± 0.2 kDa for the subunit
3258 F. Krieger et al. (Eur. J. Biochem. 269) Ó FEBS 2002
was determined by mass spectrometry (Fig. 1) and
corresponds to the size derived from SDS/PAGE and

the value calculated from the amino-acid sequence
deduced from the nucleotide sequence of the KlPDC
gene (61821 Da). Size-exclusion chromatography of the
pure enzyme revealed a single peak with a molecular mass
of about 200 kDa pointing to a tetrameric structure of the
native enzyme as the typical native state of PDC. The
N-terminus of the subunit is blocked.
The circular dichroism and fluorescence spectra of
the apoenzyme and holoenzyme of KlPDC are very
similar to those of ByPDC and ScPDC (data not shown;
[11,24,25]).
Dependence of the catalytic activity of KlPDC
on the substrate concentration
Pyruvate decarboxylase from K. lactis shows a sigmoid
dependence of the catalytic activity on the substrate
concentration (Fig. 2) like other PDC species from plant
seeds and yeasts [2,10,22,26–30]. The only known exception
is PDC from Zymomonas mobilis that displays a hyperbolic
dependence [31]. However, the sigmoidicity of the plot of
velocity vs. pyruvate concentration of KlPDC, expressed by
the ratio of the parameters A and B in Eqn (4) (Table 2), is
significantly more pronounced at substrate concentrations
below 1.5 m
M
thaninthecaseofotherPDCs.Athigh
substrate concentrations (above 100 m
M
), a weak substrate
inhibition was detected and a K
i

value of 1.2
M
was
estimated. An S
0.5
value of 1.85 m
M
was calculated
accordingtoEqn(5)atpH6.0.TheS
0.5
value increased
continuously with increasing pH (data not shown) as for
other PDC species [2,22].
Characterization of the substrate activation
behaviour of KlPDC
Substrate activation was studied by the stopped-flow
technique. A distinct lag phase in the product formation
dependent on the substrate concentration was observed
under all conditions used (Fig. 3). Progress curves were
analysed using the combined zero- and first-order function
Table 1. Purification procedure for PDC from K. lactis (starting from 60 g wet cells).
Step Total activity (U) Total protein (mg) Specific activity (UÆmg
)1
) Yield of activity (%)
Crude extract (broken cells) 4500 5400 0.8 100
Streptomycin sulfate precipitation 4300 4600 0.9 96
Ammonium sulfate precipitation 2400 1900 1.3 53
Sephacryl S200HR 1900 600 3.2 42
Resource Q 1300 80 16.3 29
Resource Phe 1000 26 38.5 22

Fig. 1. Mass spectrum (MALDI TOF) of KlPDC (0.35 mgÆmL
-1
)
confirming the molecular mass of the subunit calculated on the basis of
the nucleotide sequence. Inset, SDS/PAGE (10% acrylamide) of the
purified KlPDC indicating more than 95% homogeneity.
Fig. 2. Plot of velocity vs. pyruvate concentration of the catalysis of
KlPDC. Measurements were recorded in 0.1
M
Mes/NaOH pH 6.0 at
30 °C (circles, experimental data, solid line, fit according to the
equation vð½SÞ ¼
V
max
Á½S
2
A þBÁ½Sþ½S
2
Á
À
1þ
½S
K
I
Á
, dashed line, calculated curve
using the dissociation constants K
a
¼ 207 m
M

, K
m
¼ 0.24 m
M
,and
K
iso
¼ 0.06 drawn from the stopped-flow experiments of KlPDC
according to the equation vð½SÞ ¼
V
max
Á½S
2
K
a
ÁK
iso
ÁK
m
þ½SÁK
m
Áð1 þK
iso
Þþ½S
2
). Inset,
enlarged section at high substrate concentrations demonstrating the
substrate inhibition.
Ó FEBS 2002 Substrate activation of K. lactis pyruvate decarboxylase (Eur. J. Biochem. 269) 3259
shown in Eqn (1). The initial catalytic activity (at zero time)

was determined by calculating the ratio between the initial
and steady-state velocity of absorbance change (D
0
/D
SS
). As
illustrated in Fig. 3, KlPDC is potentially inactive in the
absence of the substrate, as found for ByPDC [30].
However, the main difference of KIPDC to all other
substrate activated PDC species analyzed so far is
manifested in the plot of k
obs
vs. the substrate concentration
(Fig. 4A). Whereas in all other cases this dependence was
found to be hyperbolic [5,16], a complex function corres-
ponding to Eqn (2) with a minimum at 1.5 m
M
pyruvate
was obtained for KlPDC. According to the values for
ByPDC given in Table 2, the calculated curve for the
dependence of k
obs
on the substrate concentration displays a
weak minimum at 0.4 m
M
pyruvate too. However, because
of the high errors of the measurements at these low substrate
concentrations, the experimental verification of this mini-
mum is difficult and it was not detected in previous studies
[5,16]. On the basis of this phenomenon the kinetic studies

of KlPDC allow a refined insight in the substrate activation
behaviour of all pyruvate decarboxylases. The K
a
and k
iso
values were derived from the plot of 1/k
obs
vs. 1/[S] at
substrate concentration above 40 m
M
(Fig. 4B). The value
k
iso
could not be determined directly at saturating substrate
concentrations because of the high K
a
value of 207 m
M
and
the greater fitting errors at high pyruvate concentrations.
The regulatory site of KlPDC shows a very low affinity for
the primary binding of the substrate (Table 2) compared to
other PDCs. The K
a
value of KlPDC for pyruvate is
twofold higher than that of ByPDC and sixfold higher than
that of PsPDC. The low affinity of the substrate to the
regulatory binding site is compensated by a fast isomeriza-
tion (k
iso

) and a high affinity of the substrate to the catalytic
centre. K
m
and k
)iso
were calculated by means of Eqns (6)
and (7). The K
m
value is about three orders of magnitude
smaller than the K
a
value. Only this special ratio of all
dissociation constants (K
m
/K
a
<K
iso
<K
a
/K
m
) allowed the
detection of a minimum in the plot of k
obs
vs. pyruvate
concentration for the first time. The low K
m
value of
KlPDC demonstrates a higher substrate affinity of the

catalytic centre compared to those of ByPDC and PsPDC.
The specificity constant k
cat
/K
m
is the highest found for
activated PDC species so far and is about 40% of that of
ZmPDC (Table 2), although the catalytic constant k
cat
of
KlPDC is fourfold lower. All constants are summarized in
Table 2. It was possible to generate a plot, which fits the
data of the observed activation rate constants (k
obs
)using
the calculated constants K
a
, K
m
, k
iso
,andk
–iso
according to
Eqn (2) (solid line in Fig. 4A). Moreover, a calculated plot
Fig. 3. Stopped-flow progress curves of the catalysis of KlPDC with the
substrate pyruvate. Measurements were carried out (from top to bottom)
at 1, 2 and 10 m
M
pyruvate (at 340 nm and 25 lgKIPDCÆmL

)1
), and
at 100 and 250 m
M
pyruvate (at 366 nm and 50 lgKIPDCÆmL
)1
)in
0.1
M
Mes/NaOH pH 6.0 at 30 °C without effectors.
Table 2. Comparison of dissociation and rate constants for the catalytic mechanism of PDC from K. lactis, brewer’s yeast, Pisum sativum,and
Zymomonas mobilis. For all species the values A, B, and K
i
were derived from the fit of the plot of velocity vs. pyruvate concentration, K
a
and k
iso
from the linear part of the double reciprocal plot of 1/k
obs
vs. 1/[S], all others were calculated on the basis of Eqns (5)–(7). Note that A defines the
overall dissociation constant of SE
a
S, and K
a
ÆK
iso
that of SE
a
(see Scheme 1).
KlPDC ByPDC

a
PsPDC
b
ZmPDC
c
pH 6.0 6.0 6.1 6.0
T(°C) 30 25 30 30
A(m
M
)
2
2.95 ± 0.21 0.68 0.24 –
B(m
M
) 0.25 ± 0.10 0.54 0.73 –
A/B (m
M
) 11.80 ± 5.56 1.26 0.32 –
S
0.5
(m
M
) 1.85 ± 0.15 1.10 1.00
K
a
(m
M
) 207.00 ± 12.40 9.30 3.31 –
k
iso

(s
)1
) 3.03 ± 0.09 0.65 1.15 –
k
)iso
(s
)1
) 0.18 ± 0.11 0.10 0.12 –
K
iso
0.06 ± 0.04 0.16 0.11 –
K
a
ÆK
iso
(m
M
) 12.42 ± 8.67 1.49 0.36
K
m
(m
M
) 0.24 ± 0.11 0.47 0.66 0.4
k
cat
[monomer] (s
)1
) 40 ± 4.0 60 60 180
k
cat

/K
m
(s
)1
Æm
M
)1
) 167 ± 93 129 90 450
K
i
(
M
) 1.2 ± 0.06 0.2–0.3 – 0.58
a
J. Ermer (unpublished results), Alvarez et al. [6];
b
Dietrich & Ko
¨
nig [16], Mu
¨
cke et al. [2], U. Mu
¨
cke (unpublished results), A. Dietrich
(unpublished results);
c
Bringer-Meyer et al. [31].
3260 F. Krieger et al. (Eur. J. Biochem. 269) Ó FEBS 2002
according to Eqn (4) using the same constants from the
substrate activation (Fig. 2, dashed line) is in coincidence
with the fit of the experimental steady-state data of the

KlPDC catalysis (Fig. 2, solid line).
The steady-state concentrations of each enzyme state
are dependent on the substrate concentration and all
enzyme states are in equilibrium as illustrated in
Scheme 1. If this equilibrium is perturbed by rapidly
lowering the substrate concentration a relaxation in the
progress curve will be observed following a first-order
reaction [5] as demonstrated in Fig. 5. The dilution
process was carried out after a reaction time of 65 s
when the enzyme was activated to 95% at 2 m
M
pyruvate
(corresponding k
obs
¼ 0.05 s
)1
). The observed rate con-
stants of the dilution process correspond to the observed
rate constants of the substrate activation and show the
same dependence on the substrate concentration (inset of
Fig. 4A) as expected on the basis of the principle of
microscopic reversibility.
Pyruvamide, a substrate surrogate of pyruvate, activates
ByPDC without being converted [5]. It was impossible to
obtain a completely activated KlPDC, even at very high
pyruvamide concentrations (above 400 m
M
,Fig.6).This
was to be expected because of the high K
a

value of the
substrate pyruvate. The estimated K
a
value of pyruvamide
binding is 90 m
M
(inset of Fig. 6A). In contrast to the
results obtained with ByPDC, pyruvamide was found to be
a mixed type inhibitor (competitive and noncompetitive) for
KlPDC (data not shown). The S
0.5
value increased and V
max
decreased with increasing pyruvamide concentration
(Fig. 6B).
The quantitatively remarkable coincidence between the
dependence of velocity vs. pyruvate concentration and the
dependence of the activation rate constant k
obs
vs. pyruvate
concentration in terms of the proposed model strongly
points to the validity of the mechanism illustrated in
Scheme 1. Moreover, it qualifies the model for the analysis
of other enzyme variants with impaired substrate activation
behaviour.
Fig. 5. Progress curve of a dilution experiment from 2 m
M
to 0.666 m
M
pyruvate in 0.1

M
Mes/NaOH pH 6.0 and 30 °C. After reaching the
steady state (about 65 s reaction time) the test mixture was diluted
threefold and the reaction was followed until the steady state was
established again. The KlPDC concentration was 22 lgÆmL
)1
after
dilution.
Fig. 4. Dependence of the activation rate constant of KlPDC (k
obs
)on
the substrate concentration. (A) Plot of k
obs
vs. the substrate concen-
trationmeasuredin0.1
M
Mes/NaOH buffer pH 6.0, 5 m
M
TDP,
5m
M
magnesium sulfate at 25 °C (open circles, stopped flow
measurements; filled circles, dilution experiments; solid line, generated
curve according to the equation k
obs
¼
k
Àiso
ÁK
m

½SþK
m
þ
k
iso
Á½S
½SþK
a
and the
calculated values K
a
¼ 207 m
M
, K
m
¼ 0.24 m
M
,andK
iso
¼ 0.06). (B)
Lineweaver–Burk plot of the experimental data for substrate con-
centrations above 40 m
M
(line, linear regression with r
2
¼ 0.994).
Ó FEBS 2002 Substrate activation of K. lactis pyruvate decarboxylase (Eur. J. Biochem. 269) 3261
ACKNOWLEDGEMENTS
We thank Ines Eberhardt for providing the K. lactis strain JA6
(Laboratorium of Molecular Cell Biology, Biological Department,

Katholic University Leuven, Belgium, Present address: Department of
Molecular Biology, Gent University, Belgium). Klaus-Peter Ru
¨
cknagel
(Max-Planck Research Unit for Enzymology of protein folding) for
N-terminal amino-acid sequencing and Angelika Schierhorn for the
mass spectrometry measurements.
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