Journul of Gerirrol Microhiologj. ( 1985). 131, 2705-2709.
Printed in Great Brituin
2705
Inhibition and Inactivation of Glucose-phosphorylating Enzymes from
Sacchavomyces cevevisiae by D-Xylose
B y R . F E R N A N D E Z , P. H E R R E R O A N D F. M O R E N O *
Depu r tanieti to It1 t erf ucult a t iro de Bioquin-iica, Fucultad de Med icinu, Uti ire rsidncl de 0r iedo,
33071 Orieclo, S p i n
(Rewired 5 February 1985 ; rerised 30 April 198.5)
Three glucose-phosphorylating enzymes were separated from cell-free extracts of Sacchciromyces
wrcrisiu~~
by hydroxylapatite chromatography. Variations in the amounts of these enzymes in
cells growing on glucose and on ethanol showed that hexokinase PI was a constitutive enzyme,
whereas synthesis of hexokinase PI1 and glucokiniw! were regulated by the carbon source used.
Glucokinase proved to be a glucomannokinase with K , values of 0.04 mM for both glucose and
mannose. D-Xylose produced an irreversible inactivation of the three glucose-phosphorylating
enzymes depending on the presence or absence of ATP. Hexokinase PI inactivation required
ATP. while hexokinase PI1 was inactivated by D-xylose without ATP in the reaction mixture.
Glucokinase was protected by ATP from this inactivation. D-Xylose acted as a competitive
inhibitor of hexokinase PI and glucokinase and as a non-competitive inhibitor of hexokinase
PII.
INTRODUCTION
Yeasts produce three glucose-phosphorylating enzymes. Hexokinase PI and hexokinase
PI1 have ratios of phosphorylation of fructose to glucose of approximately 2.5 and 1.5,
respectively (Barnard, 1975; Gancedo et al., 1977). The third enzyme, glucokinase, does not use
fructose as a substrate (Maitra, 1970). The physiological roles of the three enzymes remain
unclear. Any one of them enables growth on glucose, and either of the two hexokinases enables
growth on fructose (Gancedo er al., 1977; Lob0 & Maitra, 1977) but very little is known about
the regulation of their levels in yeasts. Hirai et ul. (1977) reported the constitutive nature of
hexokinase and glucokinase synthesis in Cundida lipolj-tica. They also showed that in Candidu
rropicalis hexokinase was specifically induced by sugars, while glucokinase was a constitutive
enzyme. On the other hand, no changes were observed for the hexokinase isoenzymes of
Stii.c.h(rroni~.ce.~
cx)rwi.siue grown on non-fermentable carbon sources or glucose (Entian et al.,
1984).
Hexokinase PI1 seems to predominate in growth on glucose (Gancedo et al., 1977) and its
involvement in carbon catabolite repression has been proposed (Entian & Mecke, 1981). A
decrease in hexokinase PI1 activity induced by D-xylose leads to the relief of carbon catabolite
repression ot'invertase ( F e r n h d e z et ul., 1984). On the other hand, a highly specific inactivation
of yeast and animal hexokinase induced by D-XylOSe has been reported (De la Fuente et al., 1970;
Lazo & Sols, 1979) which is said to involve a potentially reversible paracatalytic phosphorylation
of the enzyme a t a serine residue (Menezes & Pudles, 1976). Xylose is a non-phosphorylatable
glucose analogue that competitively inhibits the hexokinase reaction (De la Fuente et d . ,1970).
This paper deals with the separation of the different glucose-phosphorylating enzymes from
Scircharomjws cererisiue. We have verified that the synthesis of the hexokinase PI is constitutive
and that the levels of hexokinase PI1 and glucokinase are regulated by the carbon source.
Moreover, the hexokinase isoenzymes and glucokinase are susceptible to specific inactivation
induced by D-xylose in different ways.
0001-2486 Q 1985 S G M
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 23:52:43
2706
R. F E R N ~ N D E Z , P. H E R R E R O A N D F. M O R E N O
METHODS
Mattv-icrls. Sigma reagents and enzymes were used throughout. Yeast Nitrogen Base was obtained from Difco.
HA- U 1t rogel h yd rox y lapat i te was purchased from L K B.
Yeast strain, gro\t,th t~tnctitionscrtici preparcition c!f'cellTfree e.xtracts. Succharon7yces cwwisiat. G-5 17 (CECT 1 3 17)
was inoculated into flasks with 300 ml medium containing 0.7% Yeast Nitrogen Base and 1 o< (wiv) glucose o r I
( v j v ) ethanol and incubated in a rotatory shaker at 28 OC. Growth was followed by changes in optical density at
600 nm. Yeasts were harvested from glucose cultures, when the OD,,,,, was 1.2 and the sugar concentration
reached 0.650,, (early exponential phase) or when glucose had been exhausted and the OD,,,, was 2.5 (stationary
phase). Cells were collected from ethanol cultures when the OD,,,,, was 0.8 and the carbon source concentration
reached 0.50; (early exponential phase). In all cases, the cells were washed twice with distilled water and
resuspended in 10 ml 10 mM-potassium phosphate buffer, pH 7.0.
For cell disruption, equal volumes of yeast cell suspension and glass beads 0.45 to 0.50 m m in diameter were
shaken at 4 "C for 10 min in ii Vibrogen Cell Mill homogenizer. This treatment disrupted all the cells, a s judged by
light microscopy. The glass beads were removed by washing the broken cell suspension through a coarse sintered
glass filter. Cell debris was removed by centrifugation at lO000g for 20 min. The supernatant fluid was used a s
cell-free extract.
Glucosr cine1 c~thanolcletrrminution. Glucose was determined by a glucose oxidase-perosidase coupled assay.
Ethanol was determined by following the reduction of N A D + by ethanol in the presence of alcohol dehydrogenase
(Fernlindez et al., 1984).
ing e ~ j w i r s Samples
.
from ce I 1-free ex t rac t s o b t a i n ed aft e r
'hronrcitogruphic sr~parution f glucose-p
different culture times, or grown in different carbon sources, were chromatographed on hydroxylapatite HAUltrogel columns (2.5 cm x 10 cm), equilibrated and eluted at 4 "C with 10 mM-potassium phosphate buffer.
pH 7.0. The enzymes were separated by a 400 ml linear potassium phosphate gradient, I0 to 300 mM, at pH 7 . 0 .
Fractions (5 ml) were collected in an Ultrorac fractionator. The gradient shape was checked with a conductivity
meter. The separated hexokinase isoenzymes and glucokinase were used for the inactivation and inhibition
experiments.
Enz.rntr us.sajx Glucose-phosphorylating activity was followed by measurement of glucose 6-phosphate
formation, coupling the reaction with N A D P + and an excess of glucose-6-phosphate dehydrogenase. N A D P H
formation was followed at 340 nm in a Perkin -Elmer spectrophotometer maintained at 25 "C in a medium
containing (final concentrations): 4 mM-glUcOSe; 20 mM-Tris/HCI buffer, pH 7 . 5 ; 7 mM-MgCIZ: 10 mM-ATP;
0.65 mM-NADP+; 2 units glucose-6-phosphate dehydrogenase and 10-50 pl of suitably diluted enzyme
preparation. Assay systems lacking A T P were used as blanks. When fructose was used as a substrate, glucose 6phosphate was measured by the addition of an excess of phosphoglucose isomerase. When mannose was used as a
substrate, glucose 6-phosphate was measured by the addition of an excess of phosphoglucose isomerase and
phosphomannose isomerase. The formation of 1 limo1 glucose 6-phosphate min-' was taken as 1 unit.
",
R E S U L T S A N D DISCUSSION
Three glucose-phosphorylating enzymes, with different specificities for glucose and fructose,
were separated from the cell-free extracts of Saccharomyes ceretlisiae by hydroxylapatite
chromatography (Fig. 1). Two of them, which phosphorylated fructose 2-5 and 1-5 times faster
than glucose, were identified as hexokinases PI and PII, respectively, and the third, with very
low or no fructose-phosphorylating activity, was identified as glucokinase. K,,, values with
glucose were 0. I2 mM for both hexokinases, and 0.04 M M for glucokinase. Using fructose as a
substrate, hexokinases PI and PI1 had K , values of 0-33 mM.
Chromatographic profiles similar to those in Fig. 1 were obtained from stationary phase cell
extracts; however, the percentages of hexokinase PI1 and glucokinase changed with the
physiological state of the yeasts (Table 1). In the presence of glucose, the amount of hexokinase
PI1 represented 64%of the total phosphotransferase activity whereas glucose consumption led
to a decrease of hexokinase PI1 activity to 30% of the total activity. The difference in activity
was not due to differencein recoveries, which were 90% in the three columns. The differences in
activity could have been due to differences in contaminating substances that affect the activity
since the eluted enzymes were not pure. On the other hand, the recoveries were consistent with
the total activity adsorbed to the hydroxylapatite columns.
From these results we concluded that hexokinase PI was synthesized constitutively but
synthesis of hexokinase PI1 and glucokinase were regulated by culture conditions. The specific
activity of glucokinase increased when the predominant metabolic conditions were gluconeo-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 23:52:43
Glucose-phosphor~~luting
enzymes from yeast
2707
h
.-*
>
.-
Y
V
20
30 40 50 60 70
Fraction no.
Fig. I . Separation of glucose-phosphorylating enzymes by hydroxylapatite chromatography. T h e celltree extract was obtained from a 6 h culture. The glucose concentration in the supernatant was 37 mM at
the time of harvesting. Samples containing 1 I units of glucose-phosphorylating activity were
chromatographed on a 2.5 cm x 10 cm column of HA-Ultrogel (LKB) and 9096 activity was recovered
from the column. After washing with 10 mM-potassium phosphate, the sample was eluted with a
400 ink1 linear gradient of potassium phosphate ( ~--), 10 to 300 mM, pH 7.0. Fractions ( 5 ml) were
assayed spectrophotometrically ;is described in Methods with glucose ( 0 )or with fructose (0)
as
substrate. The ratio of fructose phosphorylation to glucose phosphorylation at the peaks was 1.5
(hexokiniise P11) and 2.5 (hexokinase PI). The potassium phosphate concentrations at the peaks were
48 mM (hexokinase PII), 100 mM (hexokinase PI) and 150 mM (glucokinase). This type of enzyme
preparation w;is used throughout the work.
10
~
Table 1 . Actiiitj. qf'glucost.-phosphor~luti~2g
enzynzes of' yeasts grown under dizerent conditions
The yeast cells here grown and samples taken iis described in Methods. The cells were broken and the
cell-free extracts ( I I units for glucose early exponential culture; 28 units for glucose stationary culture
and I0 units tor ethanol culture) were applied to a hydroxylapatite HA-Ultrogel column. The
percentage of each enzyme was calculated on the basis of total glucose-phosphorylating activity applied
to the column.
G rok t h
mect 1u ni
Stage of
growth
Hexokinase PI
GI UcOSe
G!ucosc
Ethanol
E;I r 1y ex pon e 11t 1 ;i I
St'itionary
E ;i rl y ex pone n t I a 1
9
9
9
(" J
Hexokinase PI1
Glucokinase
( O 3
(" U f
64
30
31
26
60
60
genic, while that of hexokinase PI1 decreased under these conditions. The total phosphorylating
capacity of the yeasts was constant (data not shown) despite changes in the relative amount of
each phosphotransferase.
Glucokinase predominated in growth on ethanol and it was identified as a glucomannokinase.
Glucokinase was capable of phosphorylating mannose with a K , of 0.04 mM which was shown
to be a competitive inhibitor of glucose with a K, of 0.06 mM. Inclusion of other sugars in the
spectrophotometric assay in a proportion of 2 0 : l to the glucose molarity showed that
glucokinase did not phosphorylate fructose, galactose, sorbose, rhamnose, arabinose, mannitol,
sorbitol or ribose.
The 'in citro' effect of D-xylose on the three partially purified kinases was also studied.
D-Xylose inactivated the three kinases, the extent of this effect depending on the presence
or absence of ATP. The enzyme preparations from hydroxylapatite chromatography were
incubated with two concentrations of D-xylose with and without ATP. Samples were taken at
different times and assayed for glucose phosphorylation as indicated in Methods. The results
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 23:52:43
2708
F E R N ~ N D EPZ. ,H E R R E R O
R.
AND F. MORENO
CL
10
I
I
I
30
40
20
Time (min)
Fig. 2
1
50
-30 -20-10
0 10 20 30
1 /(Glucose concn, mM)
Fig. 3
Fig. 2. Time courses of the inactivation of glucose-phosphorylating enzymes by D-xylose. Enzyme
preparations, obtained as described for Fig. 1, were incubated a t 30 “C in 20 mM-Tris/HCl buffer,
pH 7.5, with 25 mM-D-XylOSe
A) or 100 mM-D-xylose (0,
m). Controls without pentose were
processed simultaneously (0,
a).Filled symbols indicate preincubation with 4.0 mM-Mg . A T P ; open
symbols indicate preincubation without Mg . ATP. Samples were taken and glucose-phosphorylating
activity was assayed as described in Methods. The results are expressed as the percentage of activity
remaining after the treatment.
(a,
Fig. 3 . lnhibition of glucose-phosphorylating enzymes by D-xylose. Enzyme preparations were
obtained as described for Fig. 1 . D-Glucose-phosphorylating activity was determined without D-xylose
(a),with 40 mM-D-xylose (0)
and with 80 mM-D-xylose (A).
obtained with hexokinase PI (Fig. 2a) are in agreement with those previously described for the
animal enzyme (Lazo & Sols, 1979). The inactivation of the enzyme depended on the presence of
ATP, suggesting that the mechanism of inactivation could be a phosphorylation of the protein
(Menezes & Pudles, 1976). On the other hand, hexokinase PI1 inactivation by D-xylose was
independent of ATP (Fig. 26) and glucokinase was inactivated by D-xylose but ATP protected
the enzyme from inactivation (Fig. 2c).
In addition to the inactivation observed, D-XylOSe acted as an inhibitor of the three kinases
(Fig. 3). D-XylOSe was shown to be a competitive inhibitor of hexokinase PI and glucokinase,
with K , values of 25 mM and 0.85 mM, respectively, and a non-competitive inhibitor of
hexokinase PI1 with a K, of 80 mM.
These results explain the apparent inactivation observed (Fig. 2c) for glucokinase in the
absence of ATP as an inhibition of the enzyme produced by D-xylose. Hexokinase PI showed an
inactivation pattern similar to the one described by Lazo & Sols (1979) for the animal
hexokinase isoenzymes. Also, in this case it is now known why the inactivation does not
approach completion. The inactivation of hexokinase PI by D-xylose was 50% under all the
metabolic conditions assayed. Neither inactivation rate nor inactivation percentage were
altered by inclusion of 20 mM-Mg . ATP during the preincubation period. Reactivation of the
glucose-phosphorylating enzymes in xylose-inactivated cells by transfer into a xylose-free,
glucose-containing medium was prevented by cycloheximide (Fig. 4). The reactivation was
therefore dependent on ‘de now’ protein synthesis. Dialysis of xylose-inactivated enzyme
preparations did not reactivate the phosphotransferase activity.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 23:52:43
Glucose-phosphorylating enzymes from yeast
I
2
4
I
6
8
Time (h)
2709
I
1
0
Fig. 4 Effect of cycloheximide on the reactivation of glucose-phosphorylating enzymes. Activity of
glucose-phosphorylating enzymes after transfer from a medium with 1 :o glucose and 300 mM-D-xylose
(0)
to a medium with 1 y o glucose and 100 pg cycloheximide ml-' (A)or a medium with 17; glucose
(A):0 , control culture. The arrow indicates the time at which cell transfers were done.
Our results suggest that the interaction of D-XylOSe with hexokinase PI1 took place at a site
other than the catalytic one (non-competitive inhibitor), which is in agreement with the results
obtained with mutants and provides evidence for hexokinase PI1 acting as a bifunctional
enzyme (Entian & Frohlich, 1984) with catalytic and regulatory properties. D-Xylose reverses
catabolite repression of invertase and inactivates hexokinase PI1 (Fernandez et a/., 1984) but it
is not clear whether this is due to a loss of catalytic activity or to changes in the protein
conformation. Further studies are needed to define the molecular mechanism of catabolite
repression in yeast.
We are grateful to Professor C. Nombela for critical readings of the manuscript. This work was supported in
part by a grant from the Comision Asesora de Investigacion Cientifica y Tecnica.
REFERENCES
BARNARD,E. A . (1975). Hexokinases from yeasts.
Mrthotis in En:?wwlogj. 42C. 6-20.
DE LA FUENTE.
G., LAGUNAS. R. & SOLS,A . (1970).
Induced fit in yeast hexokinase. European Joiirnul of
Biochcniistrj, 16, 276-233.
ENTIAN.K . L). & FROHLICH,
K . U . (1984). Saccharoniyces cererisiae mutants provide evidence of hexokinase PI1 as a bifunctional enzyme with catalytic
and regulatory domains for triggering carbon
catabolite repression. Journal qf' Bacturiofogj, 158,
29-35.
ENTIAK.
K . D. & MECKE,D. (198 I ). Genetic evidence
for a role of hexokinase isozyme PI1 in carbon
catabolite repression in J'rrt~clicir~~r~t~~c'e~
cm't.i.siw.
Joiirntil c!f Biologicol ClitwiistrJ-251. 870- 874.
ENTIAN,
K . D., FROHLICH,
K . U . & MECKE,
D. (1984).
Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomycrs
crrerisiae. Biochiniica 1'1 hiuphjpsica acta 799, I8 1 186.
F E R N ~ D ER..
Z HERRERO.
.
P.. GASCON,
S. & MORENO.
F. ( 1 984). Xylose induced decrease in hexokinase PI1
activity confers resistance to carbon catabolite
repression of invertase synthesis in Su('(.huroniI,(.e~.\
c*~rrlshergc.tisis.Archirw ot Micn)hiologj. 139. 1 39 142.
GANCEDO,
J. M.. CLIFTON,
D. 8c FRAENKEL.
D. G .
( 1977). Yeast hexokinase mutants. Journcrl of Biologicul Choniistrj~252, 4443-4444.
HIRAI,M., OHTANI.
E., TANAKA,
A . & FUKUI,
S. (1977).
Glucose-phosphorylating enzymes of Cantiidti yeasts
and their regulation in vivo. Biochiniictr P I hiophj'sim
t r c t t i 400. 3S7 366.
LAZO,P. A. & SOLS,A. (1979). Specific inactivation of
animal hexokinases by xylose ' i n vitro', ' i n situ' and 'in
rii~o'.FEBS Letters 90, 88-90.
LOBO.Z. & MAITRA.
P. K . (1977). Physiological role of
glucose-phosphorylating enzymes in Soc~t*huronij*cc~.s
wrcri.sicic~.Arc~lzirc~sof Biochi~niistrj~( i t i t / Biophjtsics
182, 639-645.
MAITRA. P. K . (1970). A glucokinase from Sacchuron i ~ w . t~cwri.sitrc.
v
JoiirnuI of Biologiiul Chrmistrj- 245.
7473- 743 1 .
MENEZES.L. C . & PUDLES,
J . (1976). Studies on the
active site of yeast hexokinase. Specific phosphorylation of ;I serine residue induced by D-xylose and
ATPMg. Europcvin Journul of Biocheniistrj~65, 4 1 47.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 23:52:43