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

Pyruvate decarboxylase from Kluyveromyces lactisAn enzyme with an extraordinary substrate activation behaviour Florian Krieger*, Michael Spinka, Ralph Golbik, Gerhard Hu¨bner and Stephan

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 mM 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 Mg2+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 Ei and triggers an isomeri-zation towards an active enzyme conformation SEa 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

Ka is the dissociation constant of the substrate binding

to the regulatory site preceding the isomerization The isomerization constant Kiso is equal to the ratio

k–iso/kiso T he Km value for substrate conversion is defined as:

Km ¼ k3 ðk2 þ k1Þ

k1 ðk2 þ k3Þ

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: koenig@biochemtech.uni-halle.de

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; T DP, thiamine

diphosphate.

*Present address: Biozentrum, Universita¨t Basel, Departement

Biophysikalische Chemie, Klingelbergstrasse 70, 4056 Basel,

Switzerland.

Note: a web site is available at http://www.biochemtech.uni-halle.de/

(Received 20 March 2002, accepted 17 May 2002)

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

M A T E R I A L S A N D M E T H O D S

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 mMthiamine and 0.1 mMmagnesium sulfate The yeast

cultures (1 L) were inoculated with precultures (10% of the

volume) and grew for at least 24 h at 29C at a shaker

frequency of 110 r.p.m

Purification of PDC fromK 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 4C Frozen K lactis cells

(60 g) were thawed in 200 mL of 100 mM sodium

phos-phate pH 6.1, 5 mM dithiothreitol, 0.1 mM TDP, 0.1 mM

magnesium sulfate, 5% (v/v) glycerol, 10 lM

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 4C 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 4C, 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 mM Mes/NaOH pH 6.0,

150 mMammonium 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 mM 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

1 mLÆmin)1) The protein was eluted using an increasing ammonium sulfate gradient (0–0.2M, 100 mL) Fractions containing KlPDC were stabilized by 1M 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 mM 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 0.1M Mes/NaOH

pH 6.0, 5 mM TDP, 5 mM magnesium sulfate at 340 nm and 30C (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 mM

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)1and 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,

1 mgÆmL)1, respectively, 900 UÆmL)1) and NADH, the other KlPDC (50–100 lgÆmL)1), 10 mMTDP, and 10 mM

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 mM After 65 s the reaction mixture was diluted manually by adding buffer containing the cofactors (0.1MMes/NaOH, pH 6.0, 5 mMTDP, 5 mMmagnesium sulfate, 45 UÆmL)1alcohol dehydrogenase and 0.3–1.0 mM

NADH) in a ratio of 1 : 2, 1 : 3 and 1 : 5 T he enzyme 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 950M )1Æcm)1 for the KlPDC subunit, derived from the amino-acid sequence using the software package of

EXPASY

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 mMMes/NaOH pH 6.0

Mass spectrometry

Homogeneous KlPDC was desalted on a HiTrap column

(5 mL) using 10 mM ammonium acetate pH 6.4 Enzyme

solution (0.7 mgÆmL)1) was mixed with an equal volume

of 90 mM 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 A0 is the initial absorbance at zero time, DSS, the

steady-state velocity of absorbance change, D0, the

corres-ponding initial velocity at zero time, and kobs, the observed

first-order rate constant of the substrate activation

A ¼ A0 DSS t þDSS D0

kobs

 ½1  expðkobs tÞ ð1Þ The zero time slopes were found to be very small in the

absence of the activator pyruvamide (D0/DSS¼ 0.018)

Inclusion of D0, 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

kobs ¼ kiso Km

½S þ Km

þ kiso ½S

½S þ Ka

ð2Þ The dissociation constants are defined in Scheme 1 The

observed rate constant of substrate activation kobs is

composed of two functions The first one, including Km,

describes a property of the catalytic centre of the enzyme,

the second, hyperbolic one, including Ka, a property of the

regulatory substrate binding site From Eqn (2), extrema of

kobscan be expected at a substrate concentration of

½Sext¼Km

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Ka=Kiso Km

p

 Ka

1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Ka=Kiso Km

In the case of Ka/Km<Kiso<Km/Ka, a maximum will occur

and in the case of Km/Ka<Kiso<Ka/Km, a minimum will

occur, otherwise [S]extwill be negative

From Scheme 1 the following steady-state rate equation

can be derived:

2

Ka Kiso Km þ ½S  Km ð1 þ KisoÞ þ ½S2

¼ Vmax ½S

2

where: Vmaxis the maximum velocity, A¼ KaÆ KisoÆ Km, the overall dissociation constant of the complex SEaS;

KaÆ Kisois the overall dissociation constant of the complex

SEa, and B¼ KmÆ (Kiso+ 1) This function follows a sigmoid shape characterized by an apparent Hill coefficient between one and two In the case of A B, the Hill coefficient is almost two, in the case of

coefficient is close to one Therefore, the larger the ratio A/B, the more pronounced the sigmoidicity of the dependence of the steady-state rate on the substrate concentration appears

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 S0.5, defining the substrate concentration at which the velocity is Vmax/2, can be obtained as

S0:5 ¼ B

2 þ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

B2

4 þ A

s

ð5Þ

The values k)isoand Kmare calculated using Eqns (6) and (7) deduced from Eqns (4) and (5):

kiso ¼ kiso A

2

Ka Kiso þ S0:5 ð1 þ KisoÞ ð7Þ These equations are valid only for enzymes without significant initial activity (D0/DSSclose to zero) This is not the case for PsPDC (D0/DSS¼ 0.25, from reference [16]) However, we have involved this enzyme species in our analysis for sake of comparison

R E S U L T S A N D D I S C U S S I O N

Purification of PDC fromK 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

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 [2,10,22,26Ờ30] 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 mM than in the case of other PDCs At high substrate concentrations (above 100 mM), a weak substrate inhibition was detected and a Ki value of 1.2 M was estimated An S0.5 value of 1.85 mM was calculated according to Eqn (5) at pH 6.0 T he S0.5 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).

Fig 1 Mass spectrum (MALDI TOF) of KlPDC (0.35 mgẳmL-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 vđơSỡ Ử V max ơS 2

A ợ BơS ợ ơS 2  

1ợ ơS

KI

 , dashed line, calculated curve using the dissociation constants K a Ử 207 m M , K m Ử 0.24 m M , and

K iso Ử 0.06 drawn from the stopped-flow experiments of KlPDC according to the equation vđơSỡ Ử Vmax ơS 2

KaKisoKmợ ơSKmđ1 ợ Kisoỡ ợ ơS 2 ) Inset, enlarged section at high substrate concentrations demonstrating the substrate inhibition.

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 (D0/DSS) 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 kobsvs 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 mM pyruvate

was obtained for KlPDC According to the values for ByPDC given in Table 2, the calculated curve for the dependence of kobson the substrate concentration displays a weak minimum at 0.4 mMpyruvate 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 Kaand kiso values were derived from the plot of 1/kobs vs 1/[S] at substrate concentration above 40 mM(Fig 4B) The value

kisocould not be determined directly at saturating substrate concentrations because of the high Kavalue of 207 mMand 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 Ka 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 (kiso) and a high affinity of the substrate to the catalytic centre Kmand k)isowere calculated by means of Eqns (6) and (7) The Kmvalue is about three orders of magnitude smaller than the Ka value Only this special ratio of all dissociation constants (Km/Ka<Kiso<Ka/Km) allowed the detection of a minimum in the plot of kobs vs pyruvate concentration for the first time The low Km value of KlPDC demonstrates a higher substrate affinity of the catalytic centre compared to those of ByPDC and PsPDC The specificity constant kcat/Km is the highest found for activated PDC species so far and is about 40% of that of ZmPDC (Table 2), although the catalytic constant kcatof 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 (kobs) using the calculated constants Ka, Km, kiso, and k–isoaccording to Eqn (2) (solid line in Fig 4A) Moreover, a calculated plot

Fig 3 Stopped-flowprogress 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 lg KIPDCÆmL)1), and

at 100 and 250 m M pyruvate (at 366 nm and 50 lg KIPDCÆmL)1) in

0.1 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).

a

J Ermer (unpublished results), Alvarez et al [6];bDietrich & Ko¨nig [16], Mu¨cke et al [2], U Mu¨cke (unpublished results), A Dietrich (unpublished results); c Bringer-Meyer et al [31].

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 mMpyruvate (corresponding kobs¼ 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 mM, Fig 6) T his was to be expected because of the high Ka value of the substrate pyruvate The estimated Kavalue of pyruvamide binding is 90 mM (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 S0.5value increased and Vmax 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 kobsvs 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)1after 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-tration measured in 0.1 M Mes/NaOH buffer pH 6.0, 5 m M TDP,

5 m M magnesium sulfate at 25 C (open circles, stopped flow

measurements; filled circles, dilution experiments; solid line, generated

curve according to the equation kobs ¼ kisoKm

½S þ K m þ kiso ½S

½S þ K a and the calculated values K a ¼ 207 m M , K m ¼ 0.24 m M , and K 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).

A C K N O W L E D G E M E N T S

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 Ruẽcknagel (Max-Planck Research Unit for Enzymology of protein folding) for N-terminal amino-acid sequencing and Angelika Schierhorn for the mass spectrometry measurements.

R E F E R E N C E S

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2 Muẽcke, U., Koẽnig, S & Huẽbner, G (1995) Purification and characterisation of PDC from pea seeds (Pisum sativum cv Miko) Biol Chem Hoppe-Seyler 376, 111Ờ117.

3 Rivoal, J., Ricard, B & Pradet, A (1990) Purification and partial characterisation of PDC from Oryza sativa L Eur J Biochem.

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4 Bianchi, M.M., Tizzani, L., Destruelle, M., Frontali, L & WeƠsolowski-Louvel, M (1996) The petite negative yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity Mol Microbiol 19, 27Ờ36.

5 Huẽbner, G., Weidhase, R & Schellenberger, A (1978) The mechanism of substrate activation of pyruvate decarboxylase: a first approach Eur J Biochem 92, 175Ờ181.

6 Alvarez, F.J., Ermer, J., Huẽbner, G., Schellenberger, A & Schowen, R.L (1991) Catalytic power of pyruvate decarboxylase Ờ rate-limiting events and microscopic rate constants from primary carbon and secondary hydrogen isotope effects J Am Chem Soc.

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12 Holzer, H., Schultz, G., Villar-Palasi, C & Juẽntgen-Sell, J (1956) Isolierung der Hefepyruvatdecarboxylase und Untersuchungen uẽber Aktivitaẽt des Enzyms in lebenden Zellen Biochem Z 327, 331Ờ344.

13 Bradford, M.M (1976) A rapid and sensitive method for the quantification of microgram of protein utilising the principle of protein-dye binding Anal Biochem 72, 248Ờ254.

14 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680Ờ685.

15 Wang, J., Golbik, R., Seliger, B., Spinka, M., Tittmann, K., Huẽbner, G & Jordan, F (2001) Consequences of a modified putative substrate-activation site on catalysis by yeast pyruvate decarboxylase Biochemistry 40, 1755Ờ1763.

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17 Breunig, K.D., Bolotin-Fukuhara, M., Bianchi, M.M., Bourgarel, D., Falcone, C., Ferrero, I., Frontali, L., Goffrini, P.,

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 lgẳmL)1 Inset, dependence of the ratio of the initial velocity of

absorbance change (D 0 ) and the steady-state velocity of absorbance

change without pyruvamide (D SS ) on the pyruvamide concentration.

(B) Plot of velocity vs pyruvate concentration recorded in 0.1 M Mes/

NaOH pH 6.0 at 30 C in the presence of pyruvamide (circles, without

pyruvamide, squares, 100 m M , triangle, 200 m M , diamond, 300 m M ,

lines, fits of the experimental data according to the equation

vđơSỡ Ử Vmax ơS 2

A ợ BơS ợ S 2 ) Inset, enlarged section at low substrate

concen-trations.

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