THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 240, No. 9, Sep embe 196.5
P in ed in U.S.A.
P ope ies and Func ion o Yeas Py u a e Ca boxylase*
M. RUIZ-AMIL, G.
DE
TORRONTEGUI, E. PALAc iN, L. CATALINA,
AND M.
LOSADA
F om he Secci6n de Fisiolog a Celula , Ins i u o de BiologZa Celula , C.S.I.C., Mad id, Spain
(Recei ed o publica ion, Feb ua y 26, 1965)
Canna a and S oppani (1, 2) ound ha bake s’ yeas con-
ains an adenosine diphospha e-dependen phosphoenolpy u a e
ca boxykinase, and hey asc ibed he essen ial physiological ole
o oxaloace a e syn hesis o i . Doub s conce ning hei in e -
p e a ion a ose, howe e , when i was e ealed (3, 4) ha cell-
ee p epa a ions o he same mic oo ganism could ca alyze he
di ec ca boxyla ion o py u a e o oxaloace a e in he p esence
o adenosine iphospha e. Yeas py u a e ca boxylase was
shown o be inhibi ed by oxala e and s imula ed by ei he co-
enzyme A o i s con e ed o m, ace yl coenzyme A. Thus,
yeas py u a e ca boxylase di e s om he a ian enzyme (5, 6),
which depends absolu ely on he p esence o ace yl coenzyme A,
and om he Aspe gillus nige (7) and Pseudomonas ci onellolis
(8) enzymes, which do no ha e any equi emen o his com-
pound. The p esen pape desc ibes a p ocedu e o pu i ica ion
o py u a e ca boxylase om bake s’ yeas , and some o he
p ope ies o his enzyme, including i s labili y, a ii y o
a ious subs a es, ac i a o s, and inhibi o s, and he e ec o
PH.
A emp s by Canna a and S oppani (1) o demons a e a
mala e enzyme in Saccha omyces ce e isiae ha e been unsuc-
cess ul. We ha e now ound, howe e , as will be epo ed in
his a icle, ha he o ma ion o his enzyme in ano he yeas ,
Rhodo o ula glu &is, depends on he p esence o malic acid o
o he ela ed compounds in he g ow h medium. Blancha d,
Ko kes, Campillo, and Ochoa (9) ha e p e iously epo ed ha
an ex emely ac i e mala e enzyme is p esen in Lac obacillus
a &inosus a e cul u ing i in he p esence o malic acid. Some
o he expe imen s included in he p esen communica ion ha e
been ca ied ou in o de o s udy he unc ion o py u a e ca -
boxylase and ela ed enzymes in bo h glycolysis and gluconeo-
genesis in yeas . Fo his pu pose, wo species ha e been g own
on di e en ca bon sou ces, and he speci ic ac i i ies o each
enzyme ha e been es ima ed in he co esponding c ude ex ac s.
The esul s showed ha py u a e ca boxylase a ied only
sligh ly, whe eas py u a e kinase, phosphoenolpy u a e ca -
boxykinase, and mala e enzyme d as ically changed acco ding
o he ca bon compound consumed by he yeas . The da a
ob ained shed new ligh on he signi icance o hese enzymes in
se e al ou s anding me abolic pa hways.
EXPERIMENTAL PROCEDURE
Yeas Species and G ow h--S. ce e isiae (bake s’ yeas ) was
acqui ed om he ma ke . R. glu inis (s ain RH-1413) and
Hansenula anomala (s ain W-10) we e kindly supplied by he
* Aided by G an AM 06848-02 om he Na ional Ins i u es o
Heal h, Uni ed S a es Public Heal h Se ice.
Resea ch Fellow o he Comisa ia de P o ec ion Escola .
Depa amen o de Fe men aciones Indus iales, C.S.I.C., Mad id,
Spain. They we e g own ae obically a 30” wi h igo ous shak-
ing in he syn he ic medium o Olson and Johnson (lo), bu wi h
one o he ollowing compounds as he only ca bon sou ce:
glucose, 2%; py u a e, 4%; mala e, 3%; aspa a e, 0.5%;
ace a e, 6%. Du ing he loga i hmic phase, he cells we e ha -
es ed by slow speed cen i uga ion and washed wi h 0.05 h
T is-HCI, pH 7.6.
P epa a ion o Cell- ee Ex ac s-The yeas c ude ex ac s
we e p epa ed in he cold by g inding esh cells in a mo a wi h
wice hei weigh o alumina and ex ac ed wi h 0.05
M
T is-
HCI, pH 7.6. A e cen i uga ion o 15 min a 15,000 x g, he
supe na an laye was used as such o he enzyme assays.
Measu emen o Enzymes-Py u a e ca boxylase was assayed
by a modi ica ion o he p ocedu e o U e and Keech (5).
Py u a e kinase (11) and mala e enzyme (12) we e de e mined
by con en ional op ical me hods. Phosphoenolpy u a e ca -
boxykinase was assayed by measu ing 14C02 ixa ion unde
essen ially he condi ions desc ibed by Canna a and S oppani
(l), excep ha lac a e dehyd ogenase and NADH we e added
o a oid in e e ence by py u a e ca boxylase (4). Enzyme
uni s a e exp essed as mic omoles o subs a e u ilized o p oduc
o med pe min.
Analy ical Me hods-NADH oxida ion and NADP educ ion
we e de e mined a oom empe a u e by measu ing he change
in abso bance a 340 np in cu e es wi h a ligh pa h o 1 cm.
14C02 ixa ion was es ima ed by measu ing he adioac i e ca bon
in s ainless s eel planche s wi h ei he a gas low o a hin window
coun e wi h e iciencies o app oxima ely 20 and 5%, espec-
i ely. P o ein was de e mined by he me hods o Low y e al.
(13) and Wa bu g and Ch is ian (14).
Ma e ialsLac a e dehyd ogenase and phospho ansace ylase
we e acqui ed om Boeh inge und Soehne. Mala e dehy-
d ogenase, glu ama e-oxaloace a e ansaminase, and py u a e
kinase we e supplied by Sigma Chemical Company.
ATP, ADP, ace ylphospha e, bio in, CoA, DEAE-cellulose,
NADH, NADP, phosphoenolpy u a e, and p o amine sul a e
we e pu chased om Sigma Chemical Company. A idin and
Sephadex G-25 we e ob ained om Nu i ional Biochemicals
Co po a ion and Pha macia, espec i ely. Alumina (A-305)
was a gi om Alcoa Company. 14C02 was acqui ed om The
Badiochemical Cen e. Ace yl-CoA was p epa ed as desc ibed
by S ad man (15). All o he chemicals used we e o analy ical
g ade.
RESULTS
P epa a ion o Py u a e Ca boxylaae-The enzyme has been
pu i ied abou 140- old wi h he c ude ex ac om bake s’
3485
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3486
Yeas Py u a e Ca boxylase
Vol. 240, No. 9
yeas , p epa ed as desc ibed unde “Expe imen al P ocedu e.”
The pu i ica ion p ocedu e summa ized in Table I was ca ied
ou as ollows. To he c ude ex ac (F ac ion I) con aining 20
mg o p o ein pe ml, a 2y0 solu ion o p o amine sul a e was
TABLE I
Pu i ica ion o bake s’ yeas pym a e ca boxylase
Enzyme assays we e ca ied ou by measu ing ‘4CO2 ixa ion
as ollows. A o al olume o 1.0 ml con ained, in addi ion o he
py u a e ca boxylase p epa a ion, 100 pmoles o T is-HCI (pH
7.6),
10
pmoles o sodium py u a e, 15 pmoles o KH14C03 (6 PC),
10 pmoles o ATP, 10 pmoles o MgCl*, 0.3 pmole o CoA, 10 pmoles
o cys eine, 4 uni s o glu ama e-oxaloace a e ansaminase, and
15 amoles o sodium glu ama e. A e incuba ion o 30 min a
30” and dep o einiza ion wi h 0.1 ml o 50yo ichlo oace ic acid,
he mix u e was cen i uged and he adioac i i y was de e mined
in an aliquo o he supe na an ac ion wi h an end window
coun e as desc ibed unde “Expe imen al P ocedu e.”
F ac ion
I. C ude ex ac ..
II. P o amine sul a e
supe na an .
III. Ammonium sul a e
p ecipi a e.
IV. Sephadex G-25 il-
a e.............
V. DEAE-cellulose
elua e
-
.-
-
” b 1
x
E
n
"
$
x
z cy
E .
U
0
To al To al
p o ein ac i i y
ME
400
88
11.6
9.0
0.37
Reco e y
%
100
96
103
77
12
-
7
-
Speci ic
ac i i y
nilliuni s/
mE
1.6
7.0
57.0
55.0
207.0
10 20 0 .05 .l .15
MiNUTES PROTEiN (mg)
275 pmoles o T is-HCl (pH 7.6)) 20 pmoles o sodium py u a e, 30
pmoles o NaHCO$, 10 Fmoles o ATP, 20 pmoles o MgC12, 0.9
pmole o CoA, 20 moles o cys eine, 0.4 pmole o NADH, 0.12
uni o mala e dehyd ogenase, and he indica ed amoun s o
py u a e ca boxylase (F ac ion V). The op ical densi y changes
we e measu ed as desc ibed unde “Expe imen al P ocedu e.”
FIG. 1 (le ). P opo ionali y o WOZ ixa ion o py u a e
ca boxylase wi h incuba ion ime. Expe imen al condi ions we e
as in he s anda d assay desc ibed in Table I. Py u a e ca -
boxylase (F ac ion V), 22 pg.
FIG. 2
( igh ).
P opo ionali y o py u a e ca boxylase ac i i y
wi h p o ein added. The assays we e ca ied ou spec opho o-
me ically. Each cu e e con ained in a inal olume o 3 ml:
added in he p opo ion o 0.1 mg o p o amine sul a e pe mg
o p o ein. A e s anding o 10 min, he suspension was cen-
i uged a 27,000 X g o 10 min, and he sedimen was dis-
ca ded. The esul ing supe na an (F ac ion II) was hen
ea ed wi h he same olume o 90% ammonium sul a e o
b ing he solu ion o a inal concen a ion o 45% sa u a ion.
The suspension was allowed o s and o 20 min and cen i uged
a 27,000
x
g o 10 min. The p o ein p ecipi a e (F ac ion
III) con ained all o he ac i i y o he o iginal ex ac and i
was qui e s able when kep in a deep eeze o a leas 2 weeks.
The p ecipi a e was dissol ed in 0.05
M
T is-HCI, pH 7.6 (in
he p opo ion o 2 ml o bu e pe 20 ml o he s a ing c ude
ex ac ), and each 2-ml po ion was passed h ough a Sephadex
G-25 column (10 cm high and 2 cm in diame e ) in o de o e-
mo e he ammonium sul a e ha was p esen . The passing
solu ion was dilu ed up o 10 ml wi h 0.05
M
T is-HCl, pH 7.6,
and ammonium sul a e was added o gi e a ixed inal concen-
a ion o 0.02
M
o p o ec he enzyme du ing he nex pu i ica-
ion s ep. This solu ion (F ac ion IV) was hen passed h ough
a DEAE-cellulose column (6 cm high and 1 cm in diame e )
which was p e iously ea ed as desc ibed by Seube and Rem-
be ge (8) and equilib a ed wi h 0.05
M
T is-HCl (pH 7.6)-
0.02
M
ammonium sul a e. To ge id o unwan ed p o ein, he
column was washed wi h 20 ml o 0.1
M
T is-HCl (pH 7.6)-0.02
M
ammonium sul a e. Py u a e ca boxylase was inally elu ed
wi h 5 ml o 0.15
M
T is-HCl (pH 7.6)-0.02
M
ammonium sul a e
(F ac ion V), and used as such o he s udy o he p ope ies o
he enzyme desc ibed below.
Py u a i
Ca boxylase
Assays--Du ing he pu i ica ion s ages,
py u a e
ca boxylase was assayed as desc ibed in Table I by
.lO
al
2
.-
E
l
a
0
4
i
d .05
a
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Sep embe 1965 M. Ruiz-Amil, G. de To on egui, E. Palacidn, L. Ca alina, and M. Losada 3487
measu ing WOZ ixa ion in he p esence o glu ama e and glu-
ama e-oxaloaee a e ansaminase (4). The p opo ionali y o
W02 ixa ion wi h he incuba ion ime is shown in Fig. 1. Py u-
a e ca boxylase was al e na i ely assayed spec opho ome i-
ally by measu ing oxaloace a e o ma ion wi h he aid o
mala e dehyd ogenase and NADH (4). A ypical spec opho o-
me ic assay showing he e ec o he amoun o enzyme on he
eac ion a e is shown in Fig. 2.
Labili y
o
Py u a e Ca boxylase-The pu i ied enzyme was
qui e uns able and i los abou 25% o i s ac i i y when kep
o 3 hou s a 22”, he inac i a ion being g ea e a 0”. A e a
&j mM 1-l
.5 1.0 1.5 2.0
[PYRUVATE] ( nM )
2 6 10
& (mM)‘’
1 2 3 4 5
[ATPI (mM 1
FIG.
3. E ec o py u a e and ATP concen a ion on py u a e
ca boxylase ac i i y. The assays we e ca ied ou spec opho o-
me ically. Each cu e e con ained in a inal olume o 3 ml:
74 pg o py u a e ca boxylase (F ac ion V), 0.24 uni o mala e
dehyd ogenase, 500 pmoles o T is-HCl (pH 8.4), 30 moles o
KHCOJ, 20 *moles o MgC12, 0.9 mole o CoA, 20 @moles o cys-
eine, 0.4 pmole o NADH, and ei he 10 pmoles o ATP and py u-
a e as indica ed o 20 pmoles o py u a e and ATP as indica ed.
In he second case, he eac ion mix u e also con ained an ATP-
egene a ing sys em composed o 0.2 uni o py u a e kinase, 3
pmoles o phosphoenolpy u a e, and
100
pmoles o KCl. The
op ical densi y changes we e measu ed as desc ibed unde “Ex-
pe imen al P ocedu e.”
TABLE
II
Z@‘ec
o
bica bona e
concen a ion on py u a e
ca boxylase
ac i i y
Expe imen al condi ions we e as in Fig. 3, excep ha 20 moles
o py u a e we e used and KHCOa was added as indica ed. All
o he cu e es we e equalized in KC o he maximal concen a ion
o KHC03 by adding KC1 (c . Fig. 4).
HCOa- added
I
Reac ion a e Calcula ed Km
?nM AO.D.a o/min
Expe imen 1
None...................
10.0..
33.3..
Expe imen 2
None...................
6.7.
33.3.
Expe imen 3
None...................
3.3.
33.3..
0.040
0.110
0.140
0.065
0.240
0.320
0.045
0.140
0.280
nm
3.0
2.4
2.8
pe iod o 24 hou s a ei he empe a u e, mo e han 80% o he
o iginal ac i i y had disappea ed (c . Re e ence 16). As shown
by U e , Keech, and Sc u on (16) o he a ian enzyme, ei he
suc ose (1
M)
o ammonium sul a e (45% sa u a ion) highly
s abilized he yeas py u a e ca boxylase.
E&e
o pH on
Py u a e Ca boxylase Ac i i y-Unde he ex-
pe imen al condi ions desc ibed in Table I, he enzyme exhibi ed
maximal ac i i y a pH 8.3.
K, Values
o
Reac ion Componen s-As shown p e iously (4)
wi h he c ude ex ac , he o ma ion o oxaloace a e om py u-
a e and CO2 ca alyzed by he pu i ied enzyme, equi ed ATP
and he p esence o Mg*. The e ec o py u a e concen a-
ion on py u a e ca boxylase ac i i y is p esen ed in Fig. 3.
By plo ing he ecip ocal o he py u a e concen a ion agains
he ecip ocal o he eac ion eloci y, a Michaelis cons an o
0.80 mM was ound o his subs a e. Fig. 3 also shows he
e ec o ATP concen a ion on py u a e ca boxylase ac i i y.
Since he enzyme p epa a ion was no comple ely ee o ATPase
ac i i y, an ATP- egene a ing sys em composed o py u a e
kinase and phosphoenolpy u a e was used in hese expe imen s.
F om he da a seen in he Linewea e -Bu k plo (Fig. 3), a
K, o 0.24 mM o ATP was calcula ed.
In o de o measu e he K, alue o bica bona e, i was
necessa y o ake in o accoun he endogenous concen a ion o
his compound in he eac ion mix u e since i could no be
emo ed a he pH used by acuum and gassing p ocedu es.
A e de e mining he eac ion a e when no bica bana e was
added and he V,,, a sa u a ion o his subs a e, he K, alue
could be calcula ed om any concen a ion o bica bona e added
and om he co esponding eloci y o he eac ion by applying
he Michaelis-Men en equa ion. F om he da a o he h ee
expe imen s p esen ed in Table II, an a e age K, alue o
bica bona e o 2.7 mM was es ima ed. F om hese da a, i can
also be calcula ed ha , unde he expe imen al condi ions used,
he endogenous bica bona e concen a ion o he eac ion mix u e
a ied be ween 0.5 and 1.1 mM. No assump ions conce ning
he ac i e species o CO2 ha e been made since he e is no e i-
dence a p esen on his poin (6).
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Yeas Py u ak Ca boxylase
Vol. 240, No. 9
0
- [K+l
O-O [Na’]
/ .-‘-
. .
/.
LO o-------o 0-
I
20
40
60
mM
60
FIG. 4. E ec o K+ and Na+ concen a ions on py u a e ca -
boxylase ac i i y. The assays we e ca ied ou by measu ing
WOZ ixa ion. The eac ion mix u e con ained in a inal olume
o 1 ml: 18 g o py u a e ca boxylase (F ac ion V), 2 uni s o
glu ama e-oxaloace a e ansaminase eed o NHd+ by ea men
wi h Sephadex G-25, and he ollowing in mic omoles: T is-HCl
(pH 8.4), 199; py u ic acid, 5; KHW03, 0.9 (3 NC); ATP (acid
o m), 2.5; MgCl , 10; CoA, 0.3; cys eine, 10; glu amic acid, 10;
KC1 o NaCl as indica ed. Incuba ion .ime was 3 min. O he
expe imen al condi ions we e as desc ibed in Table I,
excep
ha
he adioac i i y was de e mined wi h a gas low coun e .
a-o Ace yl-CoA
- CoA
A-A CoA + .l ml4 A seni e
.01.03 .l .2 .3
Concen a ion (mM)
FIG. 5 (Zej ). E ec o CoA and ace yl-CoA on py u a e ca -
boxylase ac i i y. Assays we e ca ied ou by measu ing “COZ
ixa ion. The eac ion mix u e con ained in a inal olume o 1.1
ml: py u a e ca boxylase (F ac ion V), 7 Fg; glu ama e-oxalo-
ace a e ansaminase, 2 uni s; T is-HCl (pH 8.4), 199 pmoles;
sodium py u a e, 10 pmoles; KHi’COa, 5 pmoles (8 PC); ATP, 5
pmoles; MgClz, 10 cmoles; KCl, 21 moles; sodium glu ama e, 15
pmoles; and CoA, ace yl-CoA, and po assium a seni e as indica ed.
Incuba ion ime was 15 min. O he expe imen al condi ions a e
desc ibed in Table I.
FIG. 6 ( igh ). E ec o CoA and ace yl-CoA enzyma ically
o med om ace yl-CoA and CoA, espec i ely, on py u a e
In measu ing he K,,, alue o Mg++, p ecau ions we e aken
o a oid complica ions which migh a ise owing o i s chela ion
wi h he ATP p esen in he eac ion mix u e. The e o e, he
ATP concen a ion was educed o 1 mM and he Mg++ con-
cen a ions used we e abo e his le el (cj. Re e ence 6). Unde
hese condi ions, a K, alue o Mg++ o 4.2 mM was calcula ed.
E ec o K+ and Na+ on Py u a e Ca boxylase Ac i i y-
Bloom and Johnson (7) ha e epo ed s imula ion o Aspe gillus
py u a e ca boxylase by K+. In o de o s udy he e ec o
his ion on he yeas enzyme, he comme cial alkali sal s o he
eac ion componen s we e con e ed o hei acid o ms, by
passage h ough a small Dowex 50-W column, and he con-
cen a ion o KH14C03 in he eac ion mix u e was g ea ly
educed. As can be seen in Fig. 4, K+ s imula ed 4C02 ixa ion
inc easingly wi h concen a ion whe eas Na+ inhibi ed i .
Ac i a ion o Py u a e Ca boxylase by CoA
and
Ace yl-CoA-
The p e ious inding (4) ha he py u a e ca boxylase ac i i y
was inc eased abou a- old by CoA has now been ein es iga ed
in mo e de ail wi h he pu i ied enzyme. Since he possibili y
could no be excluded ha , e en wi h he pu i ied p epa a ion,
ace yl-CoA could be o med om py u a e and CoA in ou
sys em, a seni e was added o he eac ion mix u e a concen-
a ions up o 10 imes highe han hose epo ed o inhibi he
py u a e oxida ion sys em (17, 18). I was ound ha om
0.1 mM o 0.3 mM a seni e had li le e ec on he ca boxylase
eac ion, ei he in he p esence o in he absence o CoA and
ace yl-CoA. Fig. 5 shows he e ec o inc easing concen a ions
- CoA
o-o Ace yl-CoA
.l .2 .3
Concen a ion (mM 1
ca boxylase ac i i y. Assays we e ca ied ou by measu ing
“CO, ixa ion. The eac ion mix u e con ained in a inal olume
o 1.15 ml: py u a e ca boxylase, (F ac ion V), 7 ig; glu ama e-
oxaloace a e ansaminase, 2 uni s; T is-HCl (pH 8.4), 166 pmoles;
sodium py u a e, 10 moles; KHi%Oa, 5 amoles (8 PC); ATP, 5
pmoles; MgC12, 10 pmoles; KCl, 96 pmoles; sodium glu ama e, 15
wmoles; one o he wo ollowing enzyme sys ems: O-O, ace yl-
CoA (as indica ed), po assium a sena e (45 pmoles), and phos-
pho ansace ylase (4 uni s) o gene a ing CoA om ace yl-CoA;
o O-O, CoA (as indica ed), ace ylphospha e (5 pmoles), and
phospho ansace ylase (4 uni s) o gene a ing ace yl-CoA om
CoA. O he expe imen al condi ions we e as desc ibed in Table I.
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Sep embe 1965
M. Ruiz-Amil, G. de To on egui, E. Palacidn, L. Ca alina, and M. Losada
3489
o ace yl-CoA and COA (wi h and wi hou a seni e) on py u a e
ca boxylase ac i i y, and i demons a es ha , al hough ace yl-
CoA is a mo e e ec i e ac i a o , bo ,h compounds p oduce
compa able e ec s. In o de o p o e unequi ocally ha hese
conclusions we e co ec , expe imen al condi ions we e de ised
ha could ensu e he s eady main enance o he le el o CoA
and ace yl-CoA e en a low concen a ions. Fo his pu pose,
he wo gene a ing enzyme sys ems o CoA and ace yl-CoA
desc ibed in Fig. 6 we e employed. In comple e ag eemen
wi h he esul s o Fig. 5, Fig. 6 shows ha ac i a ion is lowe
when added ace yl-CoA is con e ed in o CoA han when added
CoA is con e ed in o ace yl-CoA. The a ios o basal ac i i y
o ace yl-CoA- and CoA-s imula ed ac i i y we e ela i ely
cons an wi h di e en p epa a ions.
E$ec o
Inhibi o s-The inhibi ion by oxala e o py u a e
ca boxylase ac i i y p e iously epo ed (3, 4) has now been
e-examined wi h he pu i ied enzyme a di e en concen a ions
o he eac ion componen s. I is appa en om he da a
p esen ed in Table III ha he inhibi o y e ec o oxala e was
independen o he concen a ions o py u a e, bica bona e,
and ATP and hus co esponded o he noncompe i i e ype.
In he case o Mg++, a s onge inhibi ion was obse ed when he
concen a ion o his ca ion was educed o 1 mM and kep 5
imes lowe han ha o ATP. Owing o he chela ion o he
Mg++ wi h he ATP, he eal concen a ion o ee Mg++ in he
eac ion mix u e is e en lowe , and he supe imposed inhibi ion
obse ed unde hese condi ions can be explained by
an addi ional
emo al o Mg++ by oxala e. Fo calcula ing he enzyme-
Inhibi o cons an , much highe amoun s o Mg++ (40 pmoles)
han o ATP (10 pmoles) we e employed, and he expe imen s
we e ca ied ou a di e en concen a ions o py u a e in he
p esence and absence o oxala e (0.1 mM) as indica ed in Fig. 7.
Unde hese condi ions, a Ki alue o oxala e o 0.07 mM was
calcula ed om he plo s o ecip ocal eloci y agains ecip ocal
subs a e concen a ion.
TABLE
III
Inhibi ion by oxala e o py u a e ca boxylase ac i i y a
di e en concen a ions
o
eac ion componen s
The assays we e ca ied ou by measu ing 14C02 ixa ion. The
comple e sys em included
in a inal olume o 1 ml: 7 g o py u-
a e ca boxylase (F ac ion V), 100 pmoles o T is-HCl (pH 8.4),
5 kmoles o sodium py u a e, 10 pmoles (4 pC) o KHiW03, 5
moles o
ATP,
10 pmoles o MgCl2, 0.3 pmole o CoA, 2
uni s o
glu ama e-oxaloace a e ansaminase, and 15 pmoles o sodium
glu ama e. O he expe imen al condi ions we e as desc ibed in
Table I, excep ha he adioac i i y was de e mined wi h a gas
low coun e .
“CO2 ixed
sys em No ox&a e Oxala e
(0.1 m?d)
Inhibi ion
1. Comple e................
2. Comple e bu 1 mM
Mg++ . . . .
3. Comple e bu 0.5 nM
ATP
4. Comple e bu 0.5 mM
py u a e . .
5. Comple e bu 1 mM
HCO$- . . . . . . .
cpm x lo-~ %
47.5 26.9 54
4.8 0.5
91
25.9
12.5
52
22.0 9.5 54
12.5 5.3 58
FIG.
7. Noncompe i i e inhibi ion o py u a e ca boxylase by
oxala e. The assays we e ca ied ou spec opho ome ically .
Each cu e e con ained in a inal olume o 3 ml: py u a e ca -
boxylase (F ac ion V), 74 pg; T is-HCl (pH 8.4), 500 amoles;
KHCO,, 150 ccmoles; ATP, 10 @moles; MgCl2, 40 pmoles; mala e
dehyd ogenase, 0.24 uni ; NADH, 0.4 mole; sodium py u a e
and po assium oxala e as indica ed. The op ical densi y changes
we e measu ed as desc ibed unde “Expe imen al P ocedu e.”
TABLE IV
E ec o a idin and bio in on py u a e ca boxyhse
The assays we e ca ied ou by measu ing ‘4CO2 ixa ion as
desc ibed o Sys em 1 in Table III excep o he a idin and
bio in addi ions. Bio in was incuba ed wi h a idin o
5 min
be o e mixing wi h he enzyme sys em.
Addi ion ‘“CO ixed
cp?n x 10-a
None...................................
62.5 milliuni s o a idin.
125 milliuni s o a idin.
62.5 milliuni s o a idin + 0.5 pmole o
bio in . . . . .._____...____._........._.
125 milliuni s o a idin + 0.5 pmole o
bio in................................
0.5 Nmole o bio in.. . . .
6.3
2.1
0.7
6.5
6.5
6.9
The almos comple e inhibi ion o he 14C0 ixing eac ion by
a idin (c . Re e ence 19) and he p e en ion o his inhibi o y
e ec by p eincuba ing he a idin wi h bio in (Table IV) demon-
s a ed ha yeas py u a e ca boxylase, like hose o li e (16),
Pseudomonas (8), and Aspe gillus (7), is a bio in enzyme.
Le els o Py u a e- ela ed Enzymes in Yeas s G own wi h Dij-
je en
Ca bon Sou ces-Table V summa izes he ac i i ies o
py u a e ca boxylase, py u a e kinase, phosphoenolpy u a e
ca boxykinase, and mala e enzyme in wo yeas species g own
as ou lined unde “Expe imen al P ocedu e” in media con aining
ei he glucose, py u a e, mala e, aspa a e, o ace a e as he
only ca bon sou ce. The speci ic ac i i y o each enzyme was
de e mined in he co esponding c ude ex ac s as desc ibed
abo e, py u a e ca boxylase being assayed adiochemically.
I
can
be seen om he da a p esen ed in his able ha py u a e
a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om

3490
Yeas Py u a e Ca boxyhe Vol. 240, No. 9
TABLE V
Ac i i ies o py u a e- ela ed enzymes in yeas s g own
on di e en ca bon sou ces
Rhodo o ula glu inis
Glucose
Py u a e
Mala e
Aspa a e
Ace a e
Hansen&a anomala
Glucose
Ace a e
-
Speci ic ac i i y
14.2
44.0 167 1.3
1.3 <0.7
18.3 4.3 5.2 8.7
18.3 1.8 5.0 10.3
5.8 1.7 18.3 78.4
5.0 9.3 14.3 36.6
12.5 2.7 21.6 26.5
7.7 2.3 15.7 63.6
6.7 1.5 14.9 56.6
11.6 8.1 29.6 17.0
12.2
10.3 <0.7
1.2 6.6
7.0 43.2
20.3 358 0.1
9.3 1.25 8.2
12.0 <0.7 22.4
<0.7
<0.7
<0.7
ca boxylase did no show g ea a ia ions in esponse o he
ca bon subs a e used by he cells. By con as , py u a e
kinase, phosphoenolpy u a e ca boxykinase, and mala e enzyme
changed d as ically acco ding o he ca bon compound con-
sumed by he cells. The le el o py u a e kinase was e y
high only when glycolysis was ac i e, i.e. in cells g own on glucose.
Phosphoenolpy u a e ca boxykinase was also ound o a y
g ea ly bu in he opposi e way, being much highe when glu-
coneogenesis was ope a i e; hus, i was low in he p esence o
glucose as subs a e and inc eased la gely wi h any o he o he
ca bon sou ces. Finally, a e cul u e o he yeas s in he p es-
ence o any one o he o he subs a es, he NADP-dependen
mala e enzyme, which was always unde ec able in cells g own
on glucose, appea ed abundan ly in
Rhodo o ula
bu no in
Hansen&a. The conclusions ha can be d awn om hese
esul s will be conside ed in some de ail and compa ed wi h
hose o o he au ho s in he ollowing sec ion.
DISCUSSION
The py u a e ca boxylase desc ibed he e is appa en ly e y
simila o hose p e iously ound in a ian li e (5, 6, 16),
P.
ci onellolis
(8), and
A.
nige (7). An especially in e es ing
p ope ,y o he yeas ca boxylase is i s ac i a ion by bo h ace yl-
CoA (c . Re e ence 19) and CoA, since he chicken li e enzyme
depends absolu ely on ace yl-CoA bu no on CoA and hose o
Pseudomonas and
Aspe gillus
do no equi e ace yl-CoA. The
b ie epo by Fulle
e al.’
(20) showing ha he ATP-dependen
ca boxyla ion o py u a e in he pho osyn he ic bac e ium
Ch oma ium
(21) was s imula ed by ace yl-CoA sugges s ha
he bac e ial ca boxylase may be analogous o he yeas enzyme
om his poin o iew. The s ong noncompe i i e inhibi ion o
yeas py u a e ca boxylase by oxala e (K; = 0.07 mM) may
imply a eedback con ol mechanism since oxala e has been
shown o be he p oduc o oxaloace a e me abolism in
A.
nigs
unde ce ain cul u al condi ions (22).
Canna a and S oppani (1) concluded ha phosphoenol-
py u a e ca boxykinase seemed o be he enzyme esponsible
o ca bon dioxide ixa ion in bake s’ yeas , since hei enzyme
was inac i e in he p esence o py u a e and ATP and hei
a emp s o demons a e a mala e enzyme in cell- ee p epa a-
ions we e unsuccess ul. These conclusions a e no suppo ed
by he esul s epo ed he e which indica e ha phosphoenol-
py u a e ca boxykinase is in ol ed in he deca boxyla ion o
oxaloace a e o phosphoenolpy u a e whe eas py u a e ca -
boxylase is esponsible o he ca boxyla ion o py u a e o
oxaloace a e. Acco ding o he da a p esen ed in Table V, he
pa hways in which py u a e ca boxylase, py u a e kinase.
phosphoenolpy u a e ca boxykinase, and mala e enzyme a e
in ol ed in yeas me abolism ha e been schema ically ep e-
sen ed in Diag am 1. When glucogenesis was ac i e (cells
g own on py u a e, mala e, aspa a e, o ace a e), he le el o
py u a e kinase dec eased d as ically whe eas ha o phos-
phoenolpy u a e ca boxykinase inc eased g ea ly. This in-
dica es ha he o ma ion o phosphoenolpy u a e om any o
he compounds u ilized did no occu om py u a e h ough
he e e sal o he glycoly ic eac ion ca alyzed by py u a e
kinase, bu om oxaloace a e h ough he ac ion o phosphoenol-
py u a e ca boxykinase. These esul s a e in ag eemen wi h
he hesis ad anced by U e , Keech, and Sc u on (16) ha
py u a e kinase p obably does no play a majo ole in he syn-
hesis o phosphoenolpy u a e om py u a e du ing gluco-
neogenesis in chicken li e and wi h he obse a ions o Sh ago
e al.
(23) and La dy (24) ha phosphoenolpy u a e ca boxy-
kinase ac i i y in a li e is closely co ela ed wi h ca bohyd a e
o ma ion.
By con as , when glycolysis was ope a i e (cells g own on
glucose), he le el o py u a e kinase became e y high and ha
o phosphoenolpy u a e ca boxykinase dec eased ema kably.
This seems o indica e ha oxaloace a e was no syn hesized
om phosphoenolpy u a e by phosphoenolpy u a e ca boxy-
kinase bu a he om py u a e by py u a e ca boxylase since
unde hese g ow h condi ions no mala e enzyme was de ec able
in he cells. The e o e, py u a e seems o be he immedia e
p ecu so o oxaloace a e o bo h he ope a ion o he K ebs
cycle and he biosyn hesis o ca bon compounds. In ac , he
le el o py u a e ca boxylase eached i s maximal alue when
glucose o py u a e was used as he ca bon sou ces. An in-
c ease in py u a e ca boxylase ac i i y in a li e has been
obse ed be o e by Wagle (25) in diabe ic animals and by Hen-
ning, Sei e , and Seube (26) a e co isol adminis a ion.
Ou da a showing ha py u a e kinase is essen ial in glycolysis
bu no in gluconeogenesis a e in comple e ha mony wi h hose
jus published by K ebs and Eggles on (27). These au ho s
ound ha , on changing om a s anda d die o a low ca bo-
hyd a e die , he ac i i y o py u a e kinase in a li e ell o
abou one- hi d, and on changing o a high ca bohyd a e die ,
i ose mo e han a- old.
Wi h espec o he unc ion o he inducible mala e enzyme
i is e iden om he epo ed da a ha , when he cells we e
g own on malic acid, his enzyme was syn hesized in g ea
a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om
Sep embe 1965
M. Ruiz-Amil, G. de To on egui, E. Palacidn, L. Ca alina, and M. Losada
3491
DIAGRAM 1
Me abolic pa hways
o
py u a e in yeas
Ca bohyd a e ;::? py u a e kinase
phosphoenolpy u a e --F py u a e ---------b alanine
ace a e
amoun s, e y likely in o de di ec ly o p o ide py u a e, he
ine i able p ecu so o ace yl-CoA and alanine. Indi ec
o ma ion o py u a e wi hou he in ol emen o he mala e
enzyme is also possible h ough he combined ac ion o mala e
dehyd ogenase, phosphoenolpy u a e ca boxykinase, and py u
a e kinase. Analogously, wi h aspa a e as subs a e, py u a e
can be o med om oxaloace a e h ough mala e by mala e
dehyd ogenase and mala e enzyme o h ough phosphoenol-
py u a e by phosphoenolpy u a e ca boxykinase and py u a e
kinase. The ac ha , in epea ed expe imen s wi h aspa a e
as subs a e, mala e enzyme inc eased p opo ionally when
phosphoenolpy u a e ca boxykinase and py u a e kinase de-
c eased and ice e sa seems o sugges ha bo h pa hways
may be ope a i e. Finally, i canno ye be excluded ha
oxaloace a e may be con e ed di ec ly o py u a e since mala e
enzyme has been shown o ca alyze also he deca boxyla ion o
he o me ke oacid o py u ic acid (28).
The induc ion o mala e enzyme in
R.
glu &is when he yeas
was g own on ace a e may be explained by he accumula ion o
mala e esul ing om he ope a ion o he glyoxyla e cycle (29).
Since
H.
anomala g ew pe ec ly well on ace a e and did no
con ain unde hese condi ions de ec able amoun s o mala e
enzyme, i seems e y likely ha his enzyme is no essen ial
o gluconeogenesis in his yeas . Sh ago
e
al. (23) and La dy
e
al. (24) had p e iously concluded ha he changes in mala e
enzyme in a li e do no suppo he con en ion ha his
enzyme is di ec ly in ol ed in ca bohyd a e syn hesis om
py u a e.
The disco e y o he essen ial unc ion o he bio in-con aining
enzyme, py u a e ca boxylase, in he o ma ion o oxaloace a e
(and consequen ly o aspa a e) om py u a e in yeas may
explain why aspa ic acid can o some ex en compensa e o
bio in de iciency in yeas (30-32).
SUMMARY
Py u a e ca boxylase, he enzyme which ca alyzes he o ma-
ion o oxaloace a e om py u a e, adenosine iphospha e, and
COZ, in he p esence o Mg++, has been pu i ied om bake s’
yeas . The eac ion is s imula ed by he p esence o ace yl
ci a e
glu &a e
coenzyme A o coenzyme A and by K+ ions. The enzyme is
comple ely inhibi ed by a idin. Bio in p e en s his inhibi ion.
Oxala e has been ound o be a s ong noncompe i i e inhibi o
o he eac ion. The labili y o he enzyme, he a ini ies o he
a ious subs a es, ac i a o s and inhibi o s o he enzyme, and
he e ec o pH ha e also been in es iga ed.
The ac i i ies o py u a e ca boxylase and ela ed enzymes
ha e been de e mined in wo yeas species g own in media con-
aining di e en ca bon sou ces. Py u a e kinase was high only
when glycolysis was ac i e, whe eas phosphoenolpy u a e
ca boxykinase inc eased when gluconeogenesis was ope a i e.
Py u a e ca boxylase a ied only sligh ly wi h he ca bon com-
pound consumed by he cells. The mala e enzyme appea ed
ex emely ac i e a e cul u ing he cells in malic acid o o he
mala e p ecu so s. The essen ial unc ion o py u a e ca -
boxylase in yeas g own on glucose seems o be o p o ide oxalo-
ace a e o bo h he ope a ion o he K ebs cycle and he biosyn-
hesis o ca bon compounds.
Acknowledgmen s-We should like o hank D . A. Sols and
D . J. L. Canno as o use ul discussion and c i icism and Miss
Ma ia D. Alcain o help ul echnical assis ance.
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P ope ies and Func ion o Yeas Py u a e Ca boxylase
1965, 240:3485-3492.J. Biol. Chem.
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