ARCHIVES
OF
BIOCHEMISTRY
Physiological
AND
BIOPHYSICS
639-645
(1977)
Role of Glucose-Phosphorylating Saccharomyces cerevisiae ZITA
Tata
182,
Institute
LOB0
AND
of Fundamental
P.
Enzymes
in
K. MAITKA
Research,
Bombay
400005,
India
Received January 13, 1977 Starting with a mutant of Saccharomyces cereuisiue lacking glucokinase and both the hexokinase isozymes Pl and P2, strains were constructed, by genetic crosses, that carry single glucose-phosphorylating enzymes. The Pl and P2 isozymes and a structurally altered form of Pl hexokinase were partially purified from these strains. Hexokinases Pl, P2, and the altered Pl enzyme, respectively, phosphorylate fructose nearly four, two, and ten times as fast as they phosphorylate glucose. Strains bearing Pl show a pronounced Pasteur reaction and phosphorylate glucose, fructose, and mannose faster than those bearing the P2 isozyme. However, there is no appreciable difference between these two hexokinases in regard to the rate and the extent of growth that they sustain. The ability of yeast to grow on a particular sugar is contingent only upon the presence of an enzyme that phosphorylates it. Glucokinase seems to be responsible for catalyzing nearly half of the glucose flux in the wild type yeast. Strains bearing glucokinase alone do show a Pasteur effect.
The yeast Saccharomyces cerevisiae is known to have three enzymes for the phosphorylation of glucose; two of these are hexokinases Pl and P2 (1) known to act also on mannose and fructose, while the third enzyme glucokinase (2) does not use fructose as a substrate. The role of these enzymes in the physiology of the cell is not clear, except that glucokinase is dispensable in fructose metabolism (2). The multiplicity of these enzymes in the wild type yeast makes such a study inherently difficult. The availability of strains carrying one of these enzymes to the exclusion of the other two should help us to investigate the role of each of these enzymes in sugar metabolism. In the course of genetic studies of yeast hexokinases, we constructed strains having only a single enzyme for glucose phosphorylation. We present here the results of some of our experiments on growth and glycolytic rates of these strains. These results show that the growth of yeast on a sugar is determined merely by the presence of an enzyme that phosphorylates it, irrespective of whether it is hexokinase Pl or P2.
MATERIALS
AND METHODS
Strains. A mutant of S. cerevisiae lacking the hexokinase enzymes, 711, has been described (2). This grew on glucose because it had glucokinase. This strain was mutagenized with N-methyl-W-nitro-N-nitrosoguanidine in yeast extract-peptone medium (YEP’) containing 50 mM glucose-free galactose, and a glucokinase-negative mutant was isolated by replica plating from a YEP gala&se plate to a YEP glucose plate as a glucose-negative colony. This mutant, called 611, did not have any glucokinase or hexokinase activity, did not grow on glucose, fructose or mannose, but grew on YEP galactose medium. The genotype of the mutant, determined by independent genetic analysis, was hxkl hxk2 glkl , where hzkl and hxk2 stand for genes coding for Pl and P2 hexokinases, respectively, and glkl for the genetic determinant for glucokinase synthesis. Strains wild type for these characters used were HSC, carrying mating type a and obtained from Dr. S. N. Kakar, and X2928-3D-1C carrying the markers a ao!el trpl gall leul his2 w-a3 met14 and obtained from the Yeast Genetic Stock Center. Standard methods (3) were used in making crosses, isolating
1 Abbreviations used: YEP, yeast extract-peptone; 3P-glyceric acid, 3-phosphoglyceric acid; 39glycerate, 3-phosphoglycerate; F/G ratio, velocity of phosphorylation of fructose relative to that of glucose; DEAE, diethylaminoethyl. 639
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9661
640
LOB0
AND MAITRA
diploids, sporulation and tetrad dissection, and YEP gala&se medium was used throughout. Assay of enzymes and substrates. Fluorometric methods were employed (2) for assay using a Sargent recorder operated at 5 mV. A full-scale sensitivity of 1 nmol of NADPH was easily obtained, the limit of detection being approximately 0.015 milliunits of enzyme per milligram of protein. The effect of B-P-glyceric acid on hexokinase activity (4) was assayed in 50 mM 2-(N-morpholino) ethanesulfonic acid at pH 6.5 with 5 mM MgCl*, 0.1 mM ATP, 0.1 mM NADP, 0.5 unit of glucose 6-phosphate dehydrogenase, and 10 mM glucose; after recording an initial linear rate 1 mM 3P-glycerate was added to examine the effect of this metabolite on the enzyme activity. Fructose (30 mM) and 2 units of phosphoglucose isomerase were used in place of glucose whenever necessary. Glucose was assayed by a coupled assay using glucose oxidase and peroxidase (5). Mannose was estimated spectrophotometrically as NADPH produced by coupling the sugar phosphate with hexokinase, phosphomannose isomerase, phosphoglucase isomerase, and glucose-6-phosphate dehydrogenase; phosphomannose isomerase was omitted in the fructose assay. Protein was estimated by ultraviolet absorption (6). Chemicals. 2- (N-Morpholino) ethanesulfonic acid, fructose, mannose, and gala&se were from Sigma Chemical Co. The sugars were rendered free of contaminating glucose by incubating 1 M solutions with glucose oxidase and occasionally neutralizing the gluconic acid with dilute alkali. Glucose oxidase was from Worthington Biochemical Corporation, Glusulase was from Endo Laboratories, and other enzymes and substrates were from Boehringer. RESULTS
Construction of Strains Carrying Either of the Hexokinases Pl or P2
Genetic crosses between the hexokinase mutant 711 (hxkl hxk2 GLKl ) and the wild type strain X2923-3D-1C (HXKl HXK2 GLKl) gave strains bearing the wild type allele of either of the hexokinases Pl or P2; their genotypes were confirmed by further crosses with the hexokinase-negative strain and with tester strains carrying either the Pl or P2 enzyme. These crosses will be described elsewhere. These Pl- or PB-bearing strains were hybridized with the glucokinase mutant 611 or its derivative in the other mating type and the following diploids were obtained:
Pl strain: cx hxk2
+
+
a hxk2
hxkl
glkl
a
hxkl
+
a
hxkl
hxk.2
leul +
trpl
+
+
adel
’
P2 strain: + glkl
adel +
’
These diploids were sporulated and random spores were germinated on YlZP galactose plates; 40 segregants from each diploid were streaked on gala&se, fructose, and glucose plates. Approximately onehalf of the spores were fructose-negative and nearly one-quarter were glucose-negative as well. All the fructose-positive segregants were treated with toluene and hexokinase activity was measured in each sample using glucose and fructose. Since glucokinase does not react with fructose, the F/G ratio (velocity of phosphorylation of fructose relative to that of glucose) of hexokinase activity should be highest in segregants carrying the mutated allele of the glucokinase gene. On this basis we selected a Pl segregant having an F/G ratio nearly equal to 4 and a P2 segregant with F/G = 1.6. Both of these segregants were backcrossed with the hexokinasenegative, glucokinase-negative mutant 611 or its appropriate derivative. Sporulation of these diploids gave spore populations showing one-gene segregation for both glucose and fructose characters; this was confirmed by their growth behavior and enzyme activity. The glucose-negative haploids contained no more than 0.3% of the glucose-phosphorylating activity of the wild type. This was the mutant level of activity found in the isolate 611. The data for a representative tetrad from each of two such crosses are shown in Table I. A further backcross of these strains with the mutant 611 failed to segregate the glucose and the fructose characters, confirming that both these sugars were metabolized in these strains by the same enzyme. Growth Behavior of Pl and P2 Strains
Results in Table II summarize the features of growth of these haploid strains carrying Pl and P2 enzymes. Results for
ROLE TABLE
OF
YEAST
I
GROWTH AND ENZYME ANALYSIS OF AXOSPORES FROM TETRADS SEGREGATING EITHER Pl OR P2 GENES’ Gene segregating
Ascos ores Prom tetrad
HXKl
HXKZ
of
Growth
Sugar-phosphorylating activityb
on
Fructose
Glucase
Fructose
T6A T6B T6C T6D
+
+
+ -
+ -
1830 1.8 1110 0.6
520 1.5 358 0.4
T22A T22B T22C T22D
-
-
+ +
+ -
1.1 514 1.5 627
1.2 355 1.7 530
a Genotypes and
HXKl
+
of the diploids used were respectively
Glucase
for segregation
HXK2
a
+
+
hxk2
glkl
hxkl
a
ura.3
his2
hxk2
glkl
+
a
+
leul
hxkl
glkl
+
a
a&l
+
hxkl
glkl
kxk2
and
* Activity is expressed as milliunits/mg of protein. All four segregants from each tetrad were grown on YEP gala&se medium, and cells from the stationary phase were extracted by a French pressure cell.
TABLE
II
GROWTH
RATES OF HAPLOID STRAINS OF Saccharomyces cerevisiae CARRYING SINGLE ENZYMES OF GLUCOSE PHOSPHORYLATION~ Strain
genotype
Pl (Pl+ P2- GK-) P2 (Pl- P2+ GK-) GK (Pl- P2- GK+) Wild type (Pl+ P2+ GK+)
Doublin time (h) in YEP m eif la containing Glucase
Fructose
Mannose
Ethan01
3.1 2.5 2.0 1.7
2.8 2.5 16 1.8
3.3 2.4 3.4 1.9
3.9 3.9 3.2 3.0
a YEP medium was supplemented with 50 mM sugar or 150 mM ethanol, and the cultures were grown aerobically with shaking at 30°C. Aliquots of suspensions were diluted in 150 mM KC1 and the extinction at 650 nm was measured. Pl, P2, and GK refer, respectively, to hexokinase Pl, hexokinase P2, and glucokinase.
HEXOKINASES
641
the hexokinaseless mutant (GK strain) and the wild type are given for comparison. The doubling period was calculated from the exponential phase of growth. These experiments show that the strain bearing Pl enzyme grew at a slightly slower rate than that carrying P2 hexokinase, irrespective of what sugar they were growing on. The wild type was the fastest growing of all, while the glucokinase-bearing strain grew on glucose faster than the strains bearing either Pl or P2 enzyme. The slow growth of the glucokinase strain on fructose was due to the YEP medium alone, as it failed to grow on minimal media containing fructose as the sole carbon source. Sugar Utilization by Segregants Carrying Single Enzymes for Glucose Phosphorylation
The hexokinases Pl and P2 differ not only in the relative velocities of phosphorylation of glucose, fructose, and mannose, but also in the amounts of these enzymes per yeast cell (7). It is instructive, therefore, to examine the flux of each of these sugars that these enzymes can sustain singly. Table III gives the aerobic and anaerobic rates of sugar utilization by these strains and by a glucokinase-bearing segregant and the wild type. The Pl strain seemed to have somewhat higher anaerobic rate of utilization of all the sugars relative to the P2 strain, and had a pronounced Pasteur reaction for glucose. Of particular interest was the observation that the strain lacking the hexokinases had nearly as pronounced a Pasteur effect for glucose as the wild type. Partial Purification
ofP1 and P2 Enzymes
The properties of yeast hexokinases have so far been studied by purifying them from commercially available wild type yeast (7, 8) containing all three enzymes for phosphorylating glucose. The possibility of contamination of one preparation of enzyme with the other two cannot be ruled out. We have sought to reinvestigate the substrate specificity and the effect of 3-Pglycerate on those enzyme activities obtained from strains that carry only the
642
LOB0 TABLE
III
SUGAR UTILIZATION BY YEAST STRAINS SINGLE GLUOXE-PMXPHORYLATING
Strain
Gas phase
AND MAITRA
CARRYING ENZYME”
A
Rate of sugar utilization (~mol/min/g wet yeast)
Pl (Pl+ P2- GK-)
O2 N,
Glucase 1.4 8.1
Fructose
Mannose
5.9 7.6
3.6 6.0
P2 (I-‘- P2+ GK-)
O2 N,
2.6 3.9
1.7 2.4
1.6 3.7
GK (Pl- P2- GK+)
O2 N*
5.1 10.0
0.0 0.0
2.0 2.1
Wild type (Pl+ P2+ GK+)
OZ 10.0 5.1 3.9 20.0 12.2 2.4 N2 ’ Washed, stationary-phase cells from YEP glucose medium were suspended in 50 mM potassium phosphate buffer, pH 7.4, at 25”C, bubbled with a mixture of 5% COZ in 0, or Nz, treated with 10 to 15 mM concentrations of the sugar indicated, and aliquots were withdrawn at I-min intervals to a final concentration of 5% perchloric acid. Sugar was estimated on the neutralized supernatants. The rates were calculated from linear parts of graphs spread over at least six experimental points, covering a minimum period of 6 min. Cells were weighed wet on membrane filters.
single enzyme in question. We have also examined the properties of an altered form of Pl hexokinase synthesized by a spontaneous glucose revertant SR2 from the hexokinase-negative, glucokinase-negative mutant 611. Genetic analysis to be published elsewhere indicates that the suppressor gene in this revertant lies in the structural gene for Pl hexokinase, presumably a corrective missense mutation imposed on the mutated Pl gene giving an altered enzyme as a result. The strains PlTl3A and P2T22D carrying only Pl and P2 genes, respectively, for glucose phosphorylation, and the revertant SR2 were grown aerobically in 3 liters of YEP glucose medium at 30°C. About 25 g of washed cells from each culture was suspended in a buffer containing 50 mM potassium phosphate, pH 7.4, 2 mM EDTA, 2 mM 2-mercaptoethanol, 1 mu phenylmethanesulfonyl fluoride to pre-
vent proteolysis (9), and 3 mM glucose, and the cells were broken by a French pressure cell. The 20,000 g supernatant was subjected to precipitation with (NH&SO,. The fraction precipitating at pH 7.5 between 55 and 85% saturation of (NH&SO, had the bulk of hexokinase activity in each of the three preparations. This was dissolved in a small volume of buffer containing 10 mu potassium phosphate, pH 7.5,2 mM EDTA, 2 mM 2-mercaptoethanol, and 5 mM glucose, and dialyzed for 18 h against this buffer with frequent changes. The dialyzed enzyme solutions were absorbed on DEAE-cellulose columns and eluted with 800 ml of a linear gradient of 0 to 0.4 M KC1 prepared in the dialysis buffer used also for equilibrating and washing the columns. Two-milliliter fractions were collected. The peak of the glucose-phosphorylating activity was obtained at 110 mM KC1 in case of Pl, at 160 mM KC1 for P2, and at 145 mM KC1 for the enzyme from the revertant SR2. Fractions containing more than 1.5 units of activity were pooled and the enzymes were precipitated with 85% saturation of (NH&SO,. The pellets were dissolved in 1 ml of the buffer used for dialysis. The specific activities (units per milligram of protein) of these enzyme preparations with glucose as a substrate were as follows: 5.3 for Pl, 7.1 for P2, and 0.4 for the enzyme from the revertant, with a purification over the crude extracts of 26-fold for Pl, 21-fold for P2, and 20-fold for SR2. The F/G ratios for these preparations were 4.2, 2.3, and 10.0 for the enzymes from PlT13A, P2T22D, and SR2, respectively. Sugar Specificity
A comparison of the specificity of these hexokinase preparations towards a number of sugars is shown in Table IV. The results generally confrm previously published information on the kinetic characteristics of Pl and P2 enzymes obtained from wild type yeast (8). We wish to emphasize the somewhat higher F/G ratios of 3.9 and 1.9 for Pl and P2, respectively, as opposed to the previously published values of 3.0 and 1.3 for the enzymes prepared from the wild type yeast (8). The possibil-
ROLE OF YEAST TABLE AFFINITY
AND RELATIVE
Substrate Glucose (G) Fructose (F)
Mannose (Ml
2-Deoxyglucose (D)
VELOCITY
IV
OF GLUCOSE-PHOSPHORYLATING SUGAR@
ENZYMES
Parameter
Pl hexokinase
&I V
100
100
L V
0.94 390
1.43 190 1.9
0.1
FIG
3.9
K, V M/G
0.19 100
K, V
0.3 120 1.2
DIG
1.0
643
HEXOKINASES
P2 hexokinase 0.18
OF YEAST
SR2 revertant Pl hexokinase 0.05 100 2.4 1000 10
TOWARDS
SEVERAL
Glucokinase 0.03 100 30.0 0.4 0.0
0.1
0.1
0.1
30 0.3
280 2.8
20 0.2
0.49 80 0.8
-
1.46 45 0.4
2.26
o The Michaelis constant in mM (Km) and relative maximum velocity (V) were determined from reciprocal plots (10). V was calculated on the basis of glucose activity. For mannose and 2-deoxyglucose ADP assay was used (2); data for glucokinase are from this report. The ratios FIG, M/G, and DIG refer to the velocities of phosphorylation of fructose, mannose, and 2-deoxyglucose, respectively, relative to that of glucose. - , Not determined. * Velocity for 2-deoxyglucose was measured at 10 mM.
ity that the lower values of the F/G ratio found earlier were due to contamination of one of the hexokinase isozymes by the other, as also by glucokinase, cannot be excluded. The F/G ratio is quite useful in distinguishing a Pl enzyme from a P2 enzyme when glucokinase is absent. The revertant Pl enzyme in strain SR2 appeared substantially altered from the wild type Pl enzyme with nearly a threefold increase in the rate of fructose and mannose phosphorylation relative to glucose phosphorylation. Effect of 3-P-Glyceric
Acid
Results in Fig. 1 compare the effect of 3P-glycerate on the activity of Pl and P2 hexokinases at a limiting concentration of ATP and a relatively low pH. 3P-Glyceric acid was added after a steady-state rate of sugar phosphorylation was obtained. It was observed that Pl hexokinase showed no discernible stimulation of activity with glucose as the substrate, while P2 exhibited a fourfold stimulation of activity. With fructose, however, both the isozyme activities were stimulated, although the stimulation was more with P2 hexokinase
GLUCOSE
FIG. 1. Comparison of Pl and P2 hexokinases in regard to activation by S-P-glyceric acid. The traces refer to fluorescence of NADPH shown as a function of time. An upward deflection was caused by an increase of fluorescence, and time proceeded from right to left (scales shown in the insets). The numbers on the traces refer to rates of NADPH formation after adding 1 mM 3-P-glycerate at vertical arrows relative to the respective initial rates following the addition of the purified fractions of Pl and P2 from the DEAE-cellulose chromatography (initial rate taken as 100). The reaction mixture is described under Materials and Methods. The amount of Pl and P2 enzymes used was not the same in both cases.
644
LOB0
AND MAITRA
than with the Pl enzyme. Glucokinase showed no stimulation under these conditions.
the line for fructose. This agrees with the poorer affinity of both these enzymes for fructose relative to glucose.
Relationship between Sugar Utilization and Sugar-ATP 6-Phosphotransferase Activity in Strains Carrying PI, P2, or Glucokinase
DISCUSSION
Does the sugar-phosphorylating enzyme activity control the rate of sugar utilization in yeast? The different specific activities of glucose-phosphorylating enzymes in segregants carrying each of these enzymes offered an occasion to compare the enzyme activity with the rate of sugar utilization. Since the specific activity showed considerable fluctuation between strains, reflecting perhaps the divergent genotypes and the presence of glucose in the culture at the time of harvesting, an average value was calculated from a number of different segregants carrying a particular genotype for the enzyme in question. A large number of such strains was available during the course of genetic analysis. These data are summarized in Table V along with the value of the anaerobic sugar flux of such segregants. The results showed that the anaerobic rate of fructose consumption was nearly a linear function of the enzyme activity for this sugar. For glucose, the values for the wild type, the Pl, and the P2 strains seemed to form a linear group. Glucokinase did not appear to belong to this group. Further, the slope of the line for glucose was more than twice the slope of TABLE RELATIONSHIP
Strain genotype
BETWEEN
RATE OF SUGAR
The experiments described in this report were undertaken in order to throw some light on the physiological role of each of the three glucose-phosphorylating enzymes in S. cerevisiae. The hexokinaseless mutant grew on glucose but was unable to grow on fructose; the glucokinase-negative, hexokinase-negative mutant did not grow on either of these sugars, but grew on gala&se. Growth on glucose therefore requires either glucokinase or hexokinase, growth on fructose requires the latter enzyme, and growth on gala&se requires neither. Work with the segregants bearing only the Pl or P2 enzyme showed that these enzymes equip the cell nearly equally for growth on glucose, fructose, mannose, or maltose. The P2 isozyme of hexokinase elicited a slightly higher growth rate on its sugar substrates than the Pl isozyme despite a lower specific activity; this showed that sugar phosphorylation did not limit the rate of growth of these segregants. Examination of the kinetic characteristics of the sugar-phosphorylating enzymes partially purified from these strains indicated only marginal quantitative differences between previously published values (8) and this work. The affinity for glucose of the Pl and P2 enzymes was found to be nearly lo-fold higher than that for frucV UTILIZATION
Enzyme activity (unitslmg protein)” Glucose
Fructose
AND ENZYME
of
ACTIVITY
Sugar utilization rate (pmol/min/ g of wet cells)* Glucose 20.0 8.1 3.9 10.0
Fructose 12.2 7.6 2.4 0.0
HXKl HXK2 GLKl 1.3 1.9 HXKl kxk2 glkl 0.5 1.4 hxkl HXK2 glkl 0.4 0.5 0.3 0.0 hxkl hxk2 GLKl a Activity of sugar-ATP 6-phosphotransferase in crude extracts is the average value from at least five different segregants bearing Pl, P2, or glucokinase as the only glucose-phosphorylating enzyme. The range of variation in the specific activity of the glucose-phosphorylating enzymes from the various segregants was: for hexokinase Pl, 415 to 850; for hexokinase P2, 330 to 530; and for glucokinase 190 to 580 milliunits/mg of protein. b Rates of sugar utilization in anaerobic conditions are taken from Table III.
ROLE
OF
YEAST
tose, although the maximum velocity of fructose phosphorylation exceeded that of glucose phosphorylation by factors of four and two, respectively. The mutational alteration of the Pl enzyme in the revertant SR2 increased the velocity with fructose and mannose relative to glucose; the increased F/G ratio for the altered enzyme was particularly striking in view of the accompanying loss in the affinity for fructose and a gain in glucose affinity. However, glucose was seen to be utilized faster than fructose in this revertant (3.4 and 2.3 pmol/min/g of cells, respectively). The strain bearing Pl hexokinase utilized sugars faster than the P2 strain. While in the wild type the anaerobic rate of glucose or fructose utilization was nearly double the aerobic rate, this was much less in the strain carrying the P2 enzyme. The Pl strain utilized glucose anaerobically nearly six times faster than under aerobic conditions. The nature of determinants responsible for the aerobic inhibition of sugar phosphorylation is not understood from these experiments. However, the role of 3P-glyceric acid in the Pasteur effect (4) must be minor; the effect of this metabolite is more pronounced on the P2 enzyme, and yet the P2 strain showed little stimulation of glucose utilization on fermentative transition. Glucokinase strains also showed the Pasteur effect, although the enzyme activity was not stimulated by 3P-glycerate. As indicated earlier, a plot of sugar utilization rate by intact cells of strains Pl, P2, and the wild type versus the specific activity of their enzymes showed a nearly linear relationship, suggesting that the anaerobic sugar consumption was limited by the activity of sugar-phosphorylating enzymes. When the aerobic rates were plotted instead, this linearity broke down. Under aerobic conditions the rate of sugar utilization was not a linear function of the hexokinase activity, and something else limited the hexokinase reaction. Of the three enzymes, glucokinase had
645
HEXOKINASES
the lowest specific activity in crude extracts (Table V); however, it showed the highest glucose-phosphorylating activity in uivo. Assuming 60 milligrams of protein present per gram of wet yeast (ll), a rate of 1 micromole of sugar consumed by 1 gram of wet cells per minute requires approximately 20 milliunits of enzyme activity per milligram of protein. A comparison of the rates of phosphorylation of glucose with the specific activity of these enzymes indicated that glucokinase was capable of harnessing nearly 60% of its total capacity to phosphorylate glucose. Compared to this, the hexokinase Pl with the highest glucose-phosphorylating activity utilized under aerobic conditions no more than 5% of its rated capacity. Whether this difference can be attributed entirely to the higher affinity of glucokinase cannot be settled from these data. REFERENCES 1. COLOWICR, S. P. (1973) in The Enzymes (Boyer, P. D., ed.), Vol. 9, Part B, 3rd ed., pp. l-48, Academic Press, New York. 2. MAITRA, P. K.(1970) J. Bid. Chem. 245, 24232431. 3. SHERMAN, F., FINK, G. R., AND LAWRENCE, C. W. (1974) in Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 4. Kosow, D. P., AND ROSE, I. A. (1971) J. Biol. Chem. 246, 2618-2625. 5. DAHLQVIST, A. (1964)AnaZ. Biochem. 7,18-25. 6. LAYNE, E. (1951) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 447-454, Academic Press, New York. 7. WOMACR, F. C., WELCH, M. K., NIELSEN, J., AND COLOWICK, S. P. (1973) Arch. Biochem. Biophys. 158, 451-457. 8. RAMEL, A. H., RUSTUM, Y. M., JONES, J. G., AND BARNARD, E. A. (1971) Biochemistry 10, 3499-3508. 9. FAHRNEY, D. E., AND GOLD, A. M. (1963) J. Amer. Chem. Sot. 85, 997-1000. 10. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 56, 658-666. 11. MAITRA, P. K., AND LOBO, Z. (1971) J. Bio.!. Chem. 246, 475-488.
OF
BIOCHEMISTRY
Physiological
AND
BIOPHYSICS
639-645
(1977)
Role of Glucose-Phosphorylating Saccharomyces cerevisiae ZITA
Tata
182,
Institute
LOB0
AND
of Fundamental
P.
Enzymes
in
K. MAITKA
Research,
Bombay
400005,
India
Received January 13, 1977 Starting with a mutant of Saccharomyces cereuisiue lacking glucokinase and both the hexokinase isozymes Pl and P2, strains were constructed, by genetic crosses, that carry single glucose-phosphorylating enzymes. The Pl and P2 isozymes and a structurally altered form of Pl hexokinase were partially purified from these strains. Hexokinases Pl, P2, and the altered Pl enzyme, respectively, phosphorylate fructose nearly four, two, and ten times as fast as they phosphorylate glucose. Strains bearing Pl show a pronounced Pasteur reaction and phosphorylate glucose, fructose, and mannose faster than those bearing the P2 isozyme. However, there is no appreciable difference between these two hexokinases in regard to the rate and the extent of growth that they sustain. The ability of yeast to grow on a particular sugar is contingent only upon the presence of an enzyme that phosphorylates it. Glucokinase seems to be responsible for catalyzing nearly half of the glucose flux in the wild type yeast. Strains bearing glucokinase alone do show a Pasteur effect.
The yeast Saccharomyces cerevisiae is known to have three enzymes for the phosphorylation of glucose; two of these are hexokinases Pl and P2 (1) known to act also on mannose and fructose, while the third enzyme glucokinase (2) does not use fructose as a substrate. The role of these enzymes in the physiology of the cell is not clear, except that glucokinase is dispensable in fructose metabolism (2). The multiplicity of these enzymes in the wild type yeast makes such a study inherently difficult. The availability of strains carrying one of these enzymes to the exclusion of the other two should help us to investigate the role of each of these enzymes in sugar metabolism. In the course of genetic studies of yeast hexokinases, we constructed strains having only a single enzyme for glucose phosphorylation. We present here the results of some of our experiments on growth and glycolytic rates of these strains. These results show that the growth of yeast on a sugar is determined merely by the presence of an enzyme that phosphorylates it, irrespective of whether it is hexokinase Pl or P2.
MATERIALS
AND METHODS
Strains. A mutant of S. cerevisiae lacking the hexokinase enzymes, 711, has been described (2). This grew on glucose because it had glucokinase. This strain was mutagenized with N-methyl-W-nitro-N-nitrosoguanidine in yeast extract-peptone medium (YEP’) containing 50 mM glucose-free galactose, and a glucokinase-negative mutant was isolated by replica plating from a YEP gala&se plate to a YEP glucose plate as a glucose-negative colony. This mutant, called 611, did not have any glucokinase or hexokinase activity, did not grow on glucose, fructose or mannose, but grew on YEP galactose medium. The genotype of the mutant, determined by independent genetic analysis, was hxkl hxk2 glkl , where hzkl and hxk2 stand for genes coding for Pl and P2 hexokinases, respectively, and glkl for the genetic determinant for glucokinase synthesis. Strains wild type for these characters used were HSC, carrying mating type a and obtained from Dr. S. N. Kakar, and X2928-3D-1C carrying the markers a ao!el trpl gall leul his2 w-a3 met14 and obtained from the Yeast Genetic Stock Center. Standard methods (3) were used in making crosses, isolating
1 Abbreviations used: YEP, yeast extract-peptone; 3P-glyceric acid, 3-phosphoglyceric acid; 39glycerate, 3-phosphoglycerate; F/G ratio, velocity of phosphorylation of fructose relative to that of glucose; DEAE, diethylaminoethyl. 639
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9661
640
LOB0
AND MAITRA
diploids, sporulation and tetrad dissection, and YEP gala&se medium was used throughout. Assay of enzymes and substrates. Fluorometric methods were employed (2) for assay using a Sargent recorder operated at 5 mV. A full-scale sensitivity of 1 nmol of NADPH was easily obtained, the limit of detection being approximately 0.015 milliunits of enzyme per milligram of protein. The effect of B-P-glyceric acid on hexokinase activity (4) was assayed in 50 mM 2-(N-morpholino) ethanesulfonic acid at pH 6.5 with 5 mM MgCl*, 0.1 mM ATP, 0.1 mM NADP, 0.5 unit of glucose 6-phosphate dehydrogenase, and 10 mM glucose; after recording an initial linear rate 1 mM 3P-glycerate was added to examine the effect of this metabolite on the enzyme activity. Fructose (30 mM) and 2 units of phosphoglucose isomerase were used in place of glucose whenever necessary. Glucose was assayed by a coupled assay using glucose oxidase and peroxidase (5). Mannose was estimated spectrophotometrically as NADPH produced by coupling the sugar phosphate with hexokinase, phosphomannose isomerase, phosphoglucase isomerase, and glucose-6-phosphate dehydrogenase; phosphomannose isomerase was omitted in the fructose assay. Protein was estimated by ultraviolet absorption (6). Chemicals. 2- (N-Morpholino) ethanesulfonic acid, fructose, mannose, and gala&se were from Sigma Chemical Co. The sugars were rendered free of contaminating glucose by incubating 1 M solutions with glucose oxidase and occasionally neutralizing the gluconic acid with dilute alkali. Glucose oxidase was from Worthington Biochemical Corporation, Glusulase was from Endo Laboratories, and other enzymes and substrates were from Boehringer. RESULTS
Construction of Strains Carrying Either of the Hexokinases Pl or P2
Genetic crosses between the hexokinase mutant 711 (hxkl hxk2 GLKl ) and the wild type strain X2923-3D-1C (HXKl HXK2 GLKl) gave strains bearing the wild type allele of either of the hexokinases Pl or P2; their genotypes were confirmed by further crosses with the hexokinase-negative strain and with tester strains carrying either the Pl or P2 enzyme. These crosses will be described elsewhere. These Pl- or PB-bearing strains were hybridized with the glucokinase mutant 611 or its derivative in the other mating type and the following diploids were obtained:
Pl strain: cx hxk2
+
+
a hxk2
hxkl
glkl
a
hxkl
+
a
hxkl
hxk.2
leul +
trpl
+
+
adel
’
P2 strain: + glkl
adel +
’
These diploids were sporulated and random spores were germinated on YlZP galactose plates; 40 segregants from each diploid were streaked on gala&se, fructose, and glucose plates. Approximately onehalf of the spores were fructose-negative and nearly one-quarter were glucose-negative as well. All the fructose-positive segregants were treated with toluene and hexokinase activity was measured in each sample using glucose and fructose. Since glucokinase does not react with fructose, the F/G ratio (velocity of phosphorylation of fructose relative to that of glucose) of hexokinase activity should be highest in segregants carrying the mutated allele of the glucokinase gene. On this basis we selected a Pl segregant having an F/G ratio nearly equal to 4 and a P2 segregant with F/G = 1.6. Both of these segregants were backcrossed with the hexokinasenegative, glucokinase-negative mutant 611 or its appropriate derivative. Sporulation of these diploids gave spore populations showing one-gene segregation for both glucose and fructose characters; this was confirmed by their growth behavior and enzyme activity. The glucose-negative haploids contained no more than 0.3% of the glucose-phosphorylating activity of the wild type. This was the mutant level of activity found in the isolate 611. The data for a representative tetrad from each of two such crosses are shown in Table I. A further backcross of these strains with the mutant 611 failed to segregate the glucose and the fructose characters, confirming that both these sugars were metabolized in these strains by the same enzyme. Growth Behavior of Pl and P2 Strains
Results in Table II summarize the features of growth of these haploid strains carrying Pl and P2 enzymes. Results for
ROLE TABLE
OF
YEAST
I
GROWTH AND ENZYME ANALYSIS OF AXOSPORES FROM TETRADS SEGREGATING EITHER Pl OR P2 GENES’ Gene segregating
Ascos ores Prom tetrad
HXKl
HXKZ
of
Growth
Sugar-phosphorylating activityb
on
Fructose
Glucase
Fructose
T6A T6B T6C T6D
+
+
+ -
+ -
1830 1.8 1110 0.6
520 1.5 358 0.4
T22A T22B T22C T22D
-
-
+ +
+ -
1.1 514 1.5 627
1.2 355 1.7 530
a Genotypes and
HXKl
+
of the diploids used were respectively
Glucase
for segregation
HXK2
a
+
+
hxk2
glkl
hxkl
a
ura.3
his2
hxk2
glkl
+
a
+
leul
hxkl
glkl
+
a
a&l
+
hxkl
glkl
kxk2
and
* Activity is expressed as milliunits/mg of protein. All four segregants from each tetrad were grown on YEP gala&se medium, and cells from the stationary phase were extracted by a French pressure cell.
TABLE
II
GROWTH
RATES OF HAPLOID STRAINS OF Saccharomyces cerevisiae CARRYING SINGLE ENZYMES OF GLUCOSE PHOSPHORYLATION~ Strain
genotype
Pl (Pl+ P2- GK-) P2 (Pl- P2+ GK-) GK (Pl- P2- GK+) Wild type (Pl+ P2+ GK+)
Doublin time (h) in YEP m eif la containing Glucase
Fructose
Mannose
Ethan01
3.1 2.5 2.0 1.7
2.8 2.5 16 1.8
3.3 2.4 3.4 1.9
3.9 3.9 3.2 3.0
a YEP medium was supplemented with 50 mM sugar or 150 mM ethanol, and the cultures were grown aerobically with shaking at 30°C. Aliquots of suspensions were diluted in 150 mM KC1 and the extinction at 650 nm was measured. Pl, P2, and GK refer, respectively, to hexokinase Pl, hexokinase P2, and glucokinase.
HEXOKINASES
641
the hexokinaseless mutant (GK strain) and the wild type are given for comparison. The doubling period was calculated from the exponential phase of growth. These experiments show that the strain bearing Pl enzyme grew at a slightly slower rate than that carrying P2 hexokinase, irrespective of what sugar they were growing on. The wild type was the fastest growing of all, while the glucokinase-bearing strain grew on glucose faster than the strains bearing either Pl or P2 enzyme. The slow growth of the glucokinase strain on fructose was due to the YEP medium alone, as it failed to grow on minimal media containing fructose as the sole carbon source. Sugar Utilization by Segregants Carrying Single Enzymes for Glucose Phosphorylation
The hexokinases Pl and P2 differ not only in the relative velocities of phosphorylation of glucose, fructose, and mannose, but also in the amounts of these enzymes per yeast cell (7). It is instructive, therefore, to examine the flux of each of these sugars that these enzymes can sustain singly. Table III gives the aerobic and anaerobic rates of sugar utilization by these strains and by a glucokinase-bearing segregant and the wild type. The Pl strain seemed to have somewhat higher anaerobic rate of utilization of all the sugars relative to the P2 strain, and had a pronounced Pasteur reaction for glucose. Of particular interest was the observation that the strain lacking the hexokinases had nearly as pronounced a Pasteur effect for glucose as the wild type. Partial Purification
ofP1 and P2 Enzymes
The properties of yeast hexokinases have so far been studied by purifying them from commercially available wild type yeast (7, 8) containing all three enzymes for phosphorylating glucose. The possibility of contamination of one preparation of enzyme with the other two cannot be ruled out. We have sought to reinvestigate the substrate specificity and the effect of 3-Pglycerate on those enzyme activities obtained from strains that carry only the
642
LOB0 TABLE
III
SUGAR UTILIZATION BY YEAST STRAINS SINGLE GLUOXE-PMXPHORYLATING
Strain
Gas phase
AND MAITRA
CARRYING ENZYME”
A
Rate of sugar utilization (~mol/min/g wet yeast)
Pl (Pl+ P2- GK-)
O2 N,
Glucase 1.4 8.1
Fructose
Mannose
5.9 7.6
3.6 6.0
P2 (I-‘- P2+ GK-)
O2 N,
2.6 3.9
1.7 2.4
1.6 3.7
GK (Pl- P2- GK+)
O2 N*
5.1 10.0
0.0 0.0
2.0 2.1
Wild type (Pl+ P2+ GK+)
OZ 10.0 5.1 3.9 20.0 12.2 2.4 N2 ’ Washed, stationary-phase cells from YEP glucose medium were suspended in 50 mM potassium phosphate buffer, pH 7.4, at 25”C, bubbled with a mixture of 5% COZ in 0, or Nz, treated with 10 to 15 mM concentrations of the sugar indicated, and aliquots were withdrawn at I-min intervals to a final concentration of 5% perchloric acid. Sugar was estimated on the neutralized supernatants. The rates were calculated from linear parts of graphs spread over at least six experimental points, covering a minimum period of 6 min. Cells were weighed wet on membrane filters.
single enzyme in question. We have also examined the properties of an altered form of Pl hexokinase synthesized by a spontaneous glucose revertant SR2 from the hexokinase-negative, glucokinase-negative mutant 611. Genetic analysis to be published elsewhere indicates that the suppressor gene in this revertant lies in the structural gene for Pl hexokinase, presumably a corrective missense mutation imposed on the mutated Pl gene giving an altered enzyme as a result. The strains PlTl3A and P2T22D carrying only Pl and P2 genes, respectively, for glucose phosphorylation, and the revertant SR2 were grown aerobically in 3 liters of YEP glucose medium at 30°C. About 25 g of washed cells from each culture was suspended in a buffer containing 50 mM potassium phosphate, pH 7.4, 2 mM EDTA, 2 mM 2-mercaptoethanol, 1 mu phenylmethanesulfonyl fluoride to pre-
vent proteolysis (9), and 3 mM glucose, and the cells were broken by a French pressure cell. The 20,000 g supernatant was subjected to precipitation with (NH&SO,. The fraction precipitating at pH 7.5 between 55 and 85% saturation of (NH&SO, had the bulk of hexokinase activity in each of the three preparations. This was dissolved in a small volume of buffer containing 10 mu potassium phosphate, pH 7.5,2 mM EDTA, 2 mM 2-mercaptoethanol, and 5 mM glucose, and dialyzed for 18 h against this buffer with frequent changes. The dialyzed enzyme solutions were absorbed on DEAE-cellulose columns and eluted with 800 ml of a linear gradient of 0 to 0.4 M KC1 prepared in the dialysis buffer used also for equilibrating and washing the columns. Two-milliliter fractions were collected. The peak of the glucose-phosphorylating activity was obtained at 110 mM KC1 in case of Pl, at 160 mM KC1 for P2, and at 145 mM KC1 for the enzyme from the revertant SR2. Fractions containing more than 1.5 units of activity were pooled and the enzymes were precipitated with 85% saturation of (NH&SO,. The pellets were dissolved in 1 ml of the buffer used for dialysis. The specific activities (units per milligram of protein) of these enzyme preparations with glucose as a substrate were as follows: 5.3 for Pl, 7.1 for P2, and 0.4 for the enzyme from the revertant, with a purification over the crude extracts of 26-fold for Pl, 21-fold for P2, and 20-fold for SR2. The F/G ratios for these preparations were 4.2, 2.3, and 10.0 for the enzymes from PlT13A, P2T22D, and SR2, respectively. Sugar Specificity
A comparison of the specificity of these hexokinase preparations towards a number of sugars is shown in Table IV. The results generally confrm previously published information on the kinetic characteristics of Pl and P2 enzymes obtained from wild type yeast (8). We wish to emphasize the somewhat higher F/G ratios of 3.9 and 1.9 for Pl and P2, respectively, as opposed to the previously published values of 3.0 and 1.3 for the enzymes prepared from the wild type yeast (8). The possibil-
ROLE OF YEAST TABLE AFFINITY
AND RELATIVE
Substrate Glucose (G) Fructose (F)
Mannose (Ml
2-Deoxyglucose (D)
VELOCITY
IV
OF GLUCOSE-PHOSPHORYLATING SUGAR@
ENZYMES
Parameter
Pl hexokinase
&I V
100
100
L V
0.94 390
1.43 190 1.9
0.1
FIG
3.9
K, V M/G
0.19 100
K, V
0.3 120 1.2
DIG
1.0
643
HEXOKINASES
P2 hexokinase 0.18
OF YEAST
SR2 revertant Pl hexokinase 0.05 100 2.4 1000 10
TOWARDS
SEVERAL
Glucokinase 0.03 100 30.0 0.4 0.0
0.1
0.1
0.1
30 0.3
280 2.8
20 0.2
0.49 80 0.8
-
1.46 45 0.4
2.26
o The Michaelis constant in mM (Km) and relative maximum velocity (V) were determined from reciprocal plots (10). V was calculated on the basis of glucose activity. For mannose and 2-deoxyglucose ADP assay was used (2); data for glucokinase are from this report. The ratios FIG, M/G, and DIG refer to the velocities of phosphorylation of fructose, mannose, and 2-deoxyglucose, respectively, relative to that of glucose. - , Not determined. * Velocity for 2-deoxyglucose was measured at 10 mM.
ity that the lower values of the F/G ratio found earlier were due to contamination of one of the hexokinase isozymes by the other, as also by glucokinase, cannot be excluded. The F/G ratio is quite useful in distinguishing a Pl enzyme from a P2 enzyme when glucokinase is absent. The revertant Pl enzyme in strain SR2 appeared substantially altered from the wild type Pl enzyme with nearly a threefold increase in the rate of fructose and mannose phosphorylation relative to glucose phosphorylation. Effect of 3-P-Glyceric
Acid
Results in Fig. 1 compare the effect of 3P-glycerate on the activity of Pl and P2 hexokinases at a limiting concentration of ATP and a relatively low pH. 3P-Glyceric acid was added after a steady-state rate of sugar phosphorylation was obtained. It was observed that Pl hexokinase showed no discernible stimulation of activity with glucose as the substrate, while P2 exhibited a fourfold stimulation of activity. With fructose, however, both the isozyme activities were stimulated, although the stimulation was more with P2 hexokinase
GLUCOSE
FIG. 1. Comparison of Pl and P2 hexokinases in regard to activation by S-P-glyceric acid. The traces refer to fluorescence of NADPH shown as a function of time. An upward deflection was caused by an increase of fluorescence, and time proceeded from right to left (scales shown in the insets). The numbers on the traces refer to rates of NADPH formation after adding 1 mM 3-P-glycerate at vertical arrows relative to the respective initial rates following the addition of the purified fractions of Pl and P2 from the DEAE-cellulose chromatography (initial rate taken as 100). The reaction mixture is described under Materials and Methods. The amount of Pl and P2 enzymes used was not the same in both cases.
644
LOB0
AND MAITRA
than with the Pl enzyme. Glucokinase showed no stimulation under these conditions.
the line for fructose. This agrees with the poorer affinity of both these enzymes for fructose relative to glucose.
Relationship between Sugar Utilization and Sugar-ATP 6-Phosphotransferase Activity in Strains Carrying PI, P2, or Glucokinase
DISCUSSION
Does the sugar-phosphorylating enzyme activity control the rate of sugar utilization in yeast? The different specific activities of glucose-phosphorylating enzymes in segregants carrying each of these enzymes offered an occasion to compare the enzyme activity with the rate of sugar utilization. Since the specific activity showed considerable fluctuation between strains, reflecting perhaps the divergent genotypes and the presence of glucose in the culture at the time of harvesting, an average value was calculated from a number of different segregants carrying a particular genotype for the enzyme in question. A large number of such strains was available during the course of genetic analysis. These data are summarized in Table V along with the value of the anaerobic sugar flux of such segregants. The results showed that the anaerobic rate of fructose consumption was nearly a linear function of the enzyme activity for this sugar. For glucose, the values for the wild type, the Pl, and the P2 strains seemed to form a linear group. Glucokinase did not appear to belong to this group. Further, the slope of the line for glucose was more than twice the slope of TABLE RELATIONSHIP
Strain genotype
BETWEEN
RATE OF SUGAR
The experiments described in this report were undertaken in order to throw some light on the physiological role of each of the three glucose-phosphorylating enzymes in S. cerevisiae. The hexokinaseless mutant grew on glucose but was unable to grow on fructose; the glucokinase-negative, hexokinase-negative mutant did not grow on either of these sugars, but grew on gala&se. Growth on glucose therefore requires either glucokinase or hexokinase, growth on fructose requires the latter enzyme, and growth on gala&se requires neither. Work with the segregants bearing only the Pl or P2 enzyme showed that these enzymes equip the cell nearly equally for growth on glucose, fructose, mannose, or maltose. The P2 isozyme of hexokinase elicited a slightly higher growth rate on its sugar substrates than the Pl isozyme despite a lower specific activity; this showed that sugar phosphorylation did not limit the rate of growth of these segregants. Examination of the kinetic characteristics of the sugar-phosphorylating enzymes partially purified from these strains indicated only marginal quantitative differences between previously published values (8) and this work. The affinity for glucose of the Pl and P2 enzymes was found to be nearly lo-fold higher than that for frucV UTILIZATION
Enzyme activity (unitslmg protein)” Glucose
Fructose
AND ENZYME
of
ACTIVITY
Sugar utilization rate (pmol/min/ g of wet cells)* Glucose 20.0 8.1 3.9 10.0
Fructose 12.2 7.6 2.4 0.0
HXKl HXK2 GLKl 1.3 1.9 HXKl kxk2 glkl 0.5 1.4 hxkl HXK2 glkl 0.4 0.5 0.3 0.0 hxkl hxk2 GLKl a Activity of sugar-ATP 6-phosphotransferase in crude extracts is the average value from at least five different segregants bearing Pl, P2, or glucokinase as the only glucose-phosphorylating enzyme. The range of variation in the specific activity of the glucose-phosphorylating enzymes from the various segregants was: for hexokinase Pl, 415 to 850; for hexokinase P2, 330 to 530; and for glucokinase 190 to 580 milliunits/mg of protein. b Rates of sugar utilization in anaerobic conditions are taken from Table III.
ROLE
OF
YEAST
tose, although the maximum velocity of fructose phosphorylation exceeded that of glucose phosphorylation by factors of four and two, respectively. The mutational alteration of the Pl enzyme in the revertant SR2 increased the velocity with fructose and mannose relative to glucose; the increased F/G ratio for the altered enzyme was particularly striking in view of the accompanying loss in the affinity for fructose and a gain in glucose affinity. However, glucose was seen to be utilized faster than fructose in this revertant (3.4 and 2.3 pmol/min/g of cells, respectively). The strain bearing Pl hexokinase utilized sugars faster than the P2 strain. While in the wild type the anaerobic rate of glucose or fructose utilization was nearly double the aerobic rate, this was much less in the strain carrying the P2 enzyme. The Pl strain utilized glucose anaerobically nearly six times faster than under aerobic conditions. The nature of determinants responsible for the aerobic inhibition of sugar phosphorylation is not understood from these experiments. However, the role of 3P-glyceric acid in the Pasteur effect (4) must be minor; the effect of this metabolite is more pronounced on the P2 enzyme, and yet the P2 strain showed little stimulation of glucose utilization on fermentative transition. Glucokinase strains also showed the Pasteur effect, although the enzyme activity was not stimulated by 3P-glycerate. As indicated earlier, a plot of sugar utilization rate by intact cells of strains Pl, P2, and the wild type versus the specific activity of their enzymes showed a nearly linear relationship, suggesting that the anaerobic sugar consumption was limited by the activity of sugar-phosphorylating enzymes. When the aerobic rates were plotted instead, this linearity broke down. Under aerobic conditions the rate of sugar utilization was not a linear function of the hexokinase activity, and something else limited the hexokinase reaction. Of the three enzymes, glucokinase had
645
HEXOKINASES
the lowest specific activity in crude extracts (Table V); however, it showed the highest glucose-phosphorylating activity in uivo. Assuming 60 milligrams of protein present per gram of wet yeast (ll), a rate of 1 micromole of sugar consumed by 1 gram of wet cells per minute requires approximately 20 milliunits of enzyme activity per milligram of protein. A comparison of the rates of phosphorylation of glucose with the specific activity of these enzymes indicated that glucokinase was capable of harnessing nearly 60% of its total capacity to phosphorylate glucose. Compared to this, the hexokinase Pl with the highest glucose-phosphorylating activity utilized under aerobic conditions no more than 5% of its rated capacity. Whether this difference can be attributed entirely to the higher affinity of glucokinase cannot be settled from these data. REFERENCES 1. COLOWICR, S. P. (1973) in The Enzymes (Boyer, P. D., ed.), Vol. 9, Part B, 3rd ed., pp. l-48, Academic Press, New York. 2. MAITRA, P. K.(1970) J. Bid. Chem. 245, 24232431. 3. SHERMAN, F., FINK, G. R., AND LAWRENCE, C. W. (1974) in Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 4. Kosow, D. P., AND ROSE, I. A. (1971) J. Biol. Chem. 246, 2618-2625. 5. DAHLQVIST, A. (1964)AnaZ. Biochem. 7,18-25. 6. LAYNE, E. (1951) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 447-454, Academic Press, New York. 7. WOMACR, F. C., WELCH, M. K., NIELSEN, J., AND COLOWICK, S. P. (1973) Arch. Biochem. Biophys. 158, 451-457. 8. RAMEL, A. H., RUSTUM, Y. M., JONES, J. G., AND BARNARD, E. A. (1971) Biochemistry 10, 3499-3508. 9. FAHRNEY, D. E., AND GOLD, A. M. (1963) J. Amer. Chem. Sot. 85, 997-1000. 10. LINEWEAVER, H., AND BURK, D. (1934) J. Amer. Chem. Sot. 56, 658-666. 11. MAITRA, P. K., AND LOBO, Z. (1971) J. Bio.!. Chem. 246, 475-488.
