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-

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:

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

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

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.

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

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

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

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

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

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

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

, 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)

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

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

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

/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

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

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,

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Fig. 6. Influence of the substrate surrogate pyruvamide on the kinetics of
KlPDC. (A) Stopped-flow progress curves in the presence of 5 m
M
pyruvate and at various concentrations of pyruvamide (from bottom
to top 0, 100, 150, 200 m
M
pyruvamide) with a KlPDC concentration
of 25 lgÆmL
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. Inset, dependence of the ratio of the initial velocity of
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) and the steady-state velocity of absorbance
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