THE PURIFICATION AND PROPERTIES OF A
HEXOKINASE FROM THE CORN SCUTELLUM
HERBERT CHARLES JONES III
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
The writer wishes to exiress sincere appreciation to Dr. T. E.
Humphreys for his help, guidance, leadership, patience and for the
use of his laboratory facilities in the course of this research; to
Dr. G. Ray Noggle who inspired the writer to pursue graduate study;
to Drs. D. 8. Anthony, R. H. Biggs and T. W. Stearns for their aid and
service on the ccamittee; and to the Department of Botany and the
Department of Health, Education and Welfare for financial support
through National Defense Education Act Title IV and National
Institutes of Health Fellowships.
TABLE OF COETLTS
ACI LEDGMEI . . . . . . . . . . . .
LIST OF TABLES ........................
LIST OF FIGURES .......................
I ODUCTION .........................
REEW OF LTERATURE .....................
MATERIALS AND MEHIODS .....................
Preparation of the Enzyme
Ammonium sulfate fractionation
Absorption and elution from alumina C- gel
Assay for phosphofructokinase, phosphoglucomutase and
Chemicals and Enzymes
RESULTS ............................ 42
Nucleoside di- and triphosphates
pH and Temperature Optima
DISCUSSION .......................... 74
SIM4ARY ................... ......... 80
BIBLIOGRAPHY ................. ...... 81
BIOGRAPHICAL SKETCH . . . . . . . . . . 89
LIST OF TABLES
1. MICHALIS COSTAiTS (Ki) AMD RELATIVE M:m~ AL
RATES FOR BRAIN AD YEAST EO SE . . . . . 7
2. EFFECTS OF GLU~OE-6-P AND RELAmD COMPOUmDS O
PHOSPHORATIONS BY BRAIN HMONASE . . . .. 9
3. PUR IATION OF HEO~AE . . . . . . . 43
4. ATPASE ACTIVITY OF T~E HEmKNS PREPARATIO . . .. 5
5. suBSTRAM SPECIFICITY OF comN SCTEJIUM
EXOICEtAS ....................... 49
6. EFFECT OF MCL.EOSIDEHRIP OSFEATES AS SSUISPITw ES
FORAT~ ......................... 55
7. ACTIVATION OF EXKIASE BY MAL IOS . . . . . 60
8. EFFECT OF NUCLEOSIDE DI- AND TRIPHOSPHATS AS
INHIBITORS OF ATP IN THE EXDNASE REACT . . . 68
LIST OF FIGURES
1. Effect of glucose concentration on the rate of
phosphorylation ..................... 52
2. Lineveaver-Burk plot of the effect of glucose
concentration on the rate of phosphorylation . . 5
3. Effect of ATP concentration on the rate of
phosphorylation, and competitive inhibition
by ADP and AMP ..................... 57
4. Lineveaver-Burk plots of the effect of ATP
concentration on the rate of phosphorylation,
and competitive inhibition by ADP and AMP . . ... 59
5. Effects of Co e and Mn+H concentrations on the
rate of phosphorylation . . . . . . .... 62
6. Effect of Mg+ concentration on the rate of
phosphorylation ..................... 62
7. Lineveaver-Burk plots of the effect of PM++, Co"-
and Mnf- concentration on the rate of
phosphorylation ..................... 64
8. Lineweaver-Burk plots of the competitive inhibition
of glucose phosphorylation by xylose and D-
acetylglucosamine ................... 67
9. Effect of pH on the rate of phosphorylation
expressed as the per cent of the maximum rate
obtained ........................ 71
10. Effect of temperature on the rate of
phosphorylation ................... .. 73
The properties of plant enzymes, especially those of higher
plants, are less well known than those of similar enzymes isolated
from animal tissues. It has been assumed that the properties
associated with the characterized animal and lower plant (yeast)
enzymes are the same for higher plants. This may be the case, but
it is necessary that the plant enzymes be characterized in order to
gain a better understanding of the similarities and differences in
metabolism between plants and animals.
The enzyme, hexokinase, which catalyzes the phosphorylation of
glucose in the presence of adenosine-5'-triphosphate and magnesium ion,
occupies an important position in the metabolism of sugars in both
plants and animals. Humphreys and Garrard (47) have presented evidence
which suggests that the hexokinase reaction may be important in control-
ling the rate of glucose uptake by the corn scutellum; an organ
positioned between the root-shoot axis and endosperm of the corn seed,
where glucose absorbed from the endosperm is converted to sucrose which
is subsequently translocated to the developing seedling during germina-
tion (28). Their data indicates that glucose-6-phosphate competitively
inhibits an enzymatic step associated with net glucose uptake in
scutellum slices, and they suggest that the step might be the
hexokinase-catalyzed phosphorylation of glucose. Since it has been
demonstrated that brain hexoklnase (15, 16) and to a small extent,
yeast hexokinase (33), is inhibited by glucose-6-phosphate, although
noncompetitively, it seemed desirable to investigate the properties
of corn scutellum hexokinase.
REVIEW OF LITERATURE
In 1927, Meyerhof (73) gave the name, hexokinase, to an alcohol
precipitable fraction of autolyzed yeast which when added to extracts
of aged frog or rabbit muscle greatly enhanced glycolysls. Independ-
ently, in 1935, Euler and Adler (29) and Lutwak-Mann and Mann (66),
isolated an enzyme from yeast--heterophosphatese-which catalyzed the
following reaction in the presence of Mgr-:
Hexose + ATP~e--- -xose-6-phosphate + ADP
Mzyerhof (74), in a description of the hexokinase reaction the same
year, reported that the enzyme described by the two groups was the
same one responsible for the activity of his original preparation.
Kalckar (51), in 1939, demonstrated that kidney extracts
phosphorylated glucose and fructose and, in 1940, Geiger (36) observed
that extracts fran brain tissue phosphorylated fructose, mannose and
glucose. Belitzer and Golovskaya (5), the same year, showed that
muscle tissue contained hexokinase, which in the presence of glucose
and creatnine catalyzed the phosphorylation of glucose to hexose-6-
phosphate. In 1941, Ochoa (78) showed the presence of hexokinase in
acetone powder of rat brain. The acetone partially inactivated
adenosinetriphosphatase (ATPase). The hexokinase required Mt+ and
transferred phosphate from ATP to glucose without the liberation of
Colowick et al. (12), in 1941, demonstrated that phosphorylation
of glucose preceded the oxidation of glucose in heart muscle and
kidney extracts and that hexokinase catalyzed phosphorylation of
mannose to mannose-6-phosphate (M6P). The M6P was in turn converted
to fructose-6-phosphate (F6P) by an isomerase.
In 1946, Berger et al. (6) and Kunitz and MacDonald (57) succeeded
in crystallizing hexokinase from yeast, the only organism from which it
has been crystallized. The hexokinases from various sources have been
recently reviewed by Crane (20).
Hexokinases of Various Animal Tissues
Mammalian brain hexokinases. Colowick et al. (14) prepared
purified beef brain hexokinase by eluting acetone powder front beef
brain with water and fractional precipitation of the eluate with
aimonium sulfate. The hexokinase was precipitated between 30 and 50
per cent saturation with a two-fold increase in specific activity.
The 30 to 50 per cent fraction could be purified further by
refractionation between 45 and 50 per cent saturation with ammonium
sulfate. The product of the beef brain hexokinase reaction was shown
to be glucose-6-phosphate (G6P).
Mayerhof and Wilson (75) observed that brain extracts catalyzed
the phosphorylation of glucose and fructose at about the same rate at
0.020M concentrations of the substrates, while at concentrations of
0.0015M to 0.003M the rate was the same for glucose and much lower for
fructose. They observed the same difference relative to the ATP
concentration and there was a greater ATPase activity in the presence
of fructose than glucose. They suggested that there are actually two
separate enzymes in brain extracts--a glucokinase and a fructokinase.
Wiebelhous and Lardy (10) noted that dialyzed extracts of beef
brain showed different hexokinase activities vith respect to glucose and
fructose in the presence of inhibitory concentrations of sodium salts.
Sodium salts inhibited phosphorylation of glucose, but not of fructose,
indicating the possibility of two different hexokinases. Their beef
brain extracts promoted the phosphorylation of glucose, fructose and to
a lesser extent, mannose, but were not active with L-glucose, galactose,
L-sorbose, D-gluconate, 2-keto-0-gluconate, D-ribose, D-arabinose,
L-rhamnose and D-xylose. Inorganic pyrophosphate inhibited activity
46 to 80 per cent depending on its concentration. Magnesium ion, up
to two times the concentration of the pyrophosphate, did not reverse
inhibition. Orthophosphate was only slightly inhibitory.
Long (63) found that of five rat tissues--brain, liver, kidney,
skeletal muscle, and intestine--brain had the greatest amount of
hexokinase activity, while liver had the least.
Crane and Sole (16, 17, 86) prepared particulate hexokinase from
brain, which was free of interfering enzymes, by fractional centrifuga-
tion and solubilization of the enzyme with lipase and/or deoxycholate.
They obtained 35 per cent recovery. With this preparation, it was
found (as Weil-Malherbe and Bone (102) had reported in 1951) that G6P
inhibited hexose phosphorylation noncompetitively (15), while ADP
behaved as a competitive inhibitor (85). They (86) examined the
specificity of brain hexokinase toward thirty-five compounds structur-
ally related to glucose in order to determine which groups of the
glucose molecule were possibly involved in formation of a glucose-
brain hexokinase complex. Sixteen of the analogs served as substrates
(Table 1), five behaved as competitive inhibitors, and the remainder
were inactive. They estimated the relative influence of each hydroxyl
group of glucose by comparing the Michaelis constants (Km) and the
relative maximal rates (Vmax) of phosphorylation (Table 1). The con-
clusion was that the formation of a glucose-enzyme complex involved
the ring structure and hydroxyl groups at carbon atoms 1, 3, 4 and 6,
and that each hydroxyl group had a specific quantitative influence on
Crane and Sols (16) investigated the specificity for inhibition
of the brain hexocinase reaction by G6P and related compounds. They
tested some twenty-five compounds and only six were inhibitory
(Table 2). They concluded that the inhibitor complex involved the
pyranose ring structure, the hydroxyl groups at carbon atoms 2 and 4
and the phosphate group at carbon atom 6. They interpreted the lack
of influence by carbon atoms 1 and 3, and the influence by carbon
atom 2 as indicating that the enzyme possessed a third, specific
binding site for G6P in addition to the two for ATP and glucose and
that the data supported their hypothesis that inhibition by G6P, when
present, was a part of an intrinsic cellular mechanism for the control
of the rate of the hexokinase reaction. They found inhibition by the
hexose phosphates to be reversible, Independent of either glucose or
ATP concentration and, therefore, noncompetitive.
Maley and Lardy (67) tested the effects of a variety of N-substi-
tuted glucosamines on brain hexokinase. All were powerful inhibitors
(K-10O'3M to 10HM). They interpreted their data (and from studying
molecular models) as indicating that the substituted glucosamines
MICHAELIS CONSTAITS (Km) ARI RELATIVE MAXIMAL RATES FOR BRAIN AND
Data from references (86) and (23)
1 methyl- C-glucoside
1 methyl- F-glucoside
(TABLE 1 continued next page)
TABLE 1 (continued)
(1) (2) (3) (4) (5) (6)
Modified Brain Yeast
at Compound Km Rel. Km Rel.
Carbon (M/L) Max. (M/L) Max.
>1X10 1 0.1
+ >Lvxlo1- <0.001
2>X10U 3 <0.001
*Inhibition constant (Ki)
**Determined by competitive inhibition of fructose
***Determined at pH 7.5
EFFECT OF GLUCOSE-6-P AND RELATED COMPOUNDS ON PHOSPHORYLATIONS BY
Data from reference (16)
(1) (2) (3) (4) (5) (6)
Modified Ester Substrate Ester Per cent Ki
at concen- inhibi- (M/L)
Carbon No. traction tion
1 1,5-Sorbitan-6-P 2-Deoxyglucose 0.67 38 1X10"3
1 oC-Glucose-l,6-diP 2-Deoxyglucose 0.6 47 7X104
1 @-Glucose-1,6-diP 2-Deoxyglucose 1.0 0
1 Glucoheptulose-7-P 2-Deoxyglucose 8.6 0
1 Methyl-glucoside-6-P 2-Deoxyglucose 3.8 0
2 Mannose-6-P Mannose 8.5 0
2 2-Deoxyglucose-6-P Glucose 1.0 0
2 Glucosamine-6-P Glucose 1.5 0
2,1 Fructose-6-P 2-Deoxyglucose 2.5 0
2,1 2,5-Sorbitan-6-P 2,5-Sorbitan 13 0
2,1 Fructose-1,6-diP 2-Deoxyglucose 1.8 O
2,1 1,5-Mannitan-6-P 1,5-Mannitan 4.0 0
2,1 Mannoheptulose-7-P Mannoheptulose 2.0 0
2,1 Arabinose-5-P Arabinose 5.5 0
2,3 Altrose-6-P 2-Deoxyglucose 7.5 0
2,3,1 Ribose-5-P 2-Deoxyglucose 2.0 0
2,3,4,1 Xylose-5-P 2-Deoxyglucose 3.3 0
3 Allose-6-P 2-Deoxyglucose 9.2 58 7X103
3 3-Deoxyglucose-6-P Mannose 3.2 14 2X10-2
4 Galactose-6-P Mannose 20 0
(TABLE 2 continued next page)
TABLE 2 (continued)
No ring Gluconate-6-P
No P Glucuronate
(3) (4) (5) (6)
Substrate Ester Per cent KI
concen- inhibi- (M/L)
2-Deoxyglucose 0.45 40 7X10"4
2-Deoxyglucose 2.5 0
2-Deoxyglucose 1 0
2-Deoxyglucose 2 0
combined with the active enzyme site and that the inhibition might be
through blocking of the site on the enzyne to ATP, since the substi-
tuted groups did not overlap the carbon 6 position on the sugar.
By kinetics studies of the mechanism of the brain hexokinase
reaction, romm (31) and FroMe and Zewe (32) found that 06P acted as
a competitive inhibitor of AIP and as an uncompetitive inhibitor with
respect to glucose. Inhibition by ADP was of a more complex nature,
but appeared to be noncompetitive with respect to ATP and uncompetitive
with respect to glucose. They concluded that their results were
consistent with a mechanism involving either a phospho- or a gluco-
enzyme complex, but were at variance with any mechanism in which both
substrates need to be present simultaneously on the enzyme for the
reaction to occur. They also concluded that there seemed to be no
reason for G6P occupying a third site on the enzyme. They presented
the following compulsory pathway type mechanism for the brain bexo-
(1) Enzyme + ATP Enyme-X complex + ADP
(2) Enzym-X complex + glucose E azyme-Y complex
(3) Enzyme-Y complex --- Eaym + G6P
The nature of the Enzyme-X and Enzyme-Y complexes was unknown. The
mechanism is similar to that presented by Hamas and Kochavi (42) for
the yeast hexokinase reaction.
Kerly and Leaback (54) measured the specificity of hexokinaae of
the brain of several nonnamsmlian animals. They found that extracts
of brain from pigeon, four-day-old chick, two elasmobranchs, two
teleost fishes, frog and squid catalyzed the phosphorylation of
glucose and fructose. With pigeon brain extract, phosphorylation of
fructose was inhibited by mannose and N-acetylglucosamine, while
phosphorylation of both fructose and glucose was inhibited by G6P and
F6P. The affinity of pigeon brain hexokinase for fructose was similar
to that of the beef brain enzyme, except beef brain hexokinase was not
inhibited by F6P (16).
iMuscle. Crane and Sols (17) partially purified soluble skeletal
muscle hexokinase by fractional precipitation with cold acetone and
drying in vacuo. The enzyme was precipitated by 33 per cent acetone
(v/v) with 50 per cent recovery and a specific activity of one.
Skeletal muscle hexokinase catalyzed the phosphorylation of glucose,
mannose, fructose, glucosamine and 2-deoxy-D-glucose (2 DOG). The Km
for glucose and mannose of the muscle enzyme was about ten times
higher than that for the brain enzyme. The Km for fructose was only
slightly higher than that of brain hexokinase. Skeletal muscle
hexokinase activity was optimum at pH 8.0.
Crane and Sols (17) partially purified particulate heart muscle
hexokinase by fractional centrifugation and solubilization of the
enzyme from the particulate fraction with 0.1 per cent Triton X-100.
ADP inhibited heart muscle and skeletal muscle hexokinase competitively
(KiADP KmATP) and G6P inhibited noncompetitively. The inhibition
constants (Ki) of G6P, 1,5-sorbitan-6-P, and L-sorbose-l-P for heart
muscle hexokinase were 25 per cent, or less, of those for the brain
Strickland (90) observed that glycolysis by a muscle extract in
the presence of added hexokinase could be inhibited by 0.003M
glyceraldehyde, but the inhibition could be reversed by a small excess
of hexokinase. Be concluded that glyearaldehyde inhibition could be
pin-pointed as inhibition of glucose phosphorylation by hexokinase.
Walaas and Walaas (99) extracted an acetone powder of rat skeletal
muscle with tris-EDTA solution and obtained a five-fold increase in
specific activity by fractionating the eluate with cold ethanol. The
ethanol precipitate was dried in vacuo. Fractionation of either the
acetone powder eluate or the ethanol-precipitable fraction with
anmonium sulfate decreased recovery. EDTA, when added to the crude
hcoogenate from which the acetone powder was prepared, increased
recovery. Added glucose or ATP only slightly stabilized the ensyme.
Prolonged contact with 0.05M MgC completely inhibited the enzyme.
Chloride ion was without effect. Orthophosphate provided slight
protection and 0.03M pyrophosphate was very effective in stabilizing
muscle hexokinase. However, 0.005M pyrophosphate strongly inhibited
hexokinase during incubation of the reaction mixture. Potassium
chloride, in a narrow range of 0.02 to 0.05M, slightly activated the
hexokinase during incubation while orthophosphate inhibited. The
hexokinase had a pH optimum at 8.0 to 8.2 and one-third maximum activity
at pH 7. Maximum activity of hexokinase was observed at a molar ratio
Mg/ATP 1 for several concentrations of ATP. Magnesium ion in excess
of ATP, except at relatively high concentrations, was not inhibitory.
At a molar ratio ATP/Mg greater than 4, ATP inhibited the reaction.
The Km for both ATP and Mg was 1.7X10"3M. Inorganic pyrophosphate
inhibited the hexokinase reaction competitively with respect to MgATP
when the molar ratio was 1. At inhibitory concentrations of Mg+,
pyrophosphate had a small activating effect on the enzyme, which they
interpreted as a release of Mgs inhibition. They suggested that
inorganic pyrophosphate inhibited hexokinase by forming a Mg++-pyropbos-
phate complex which excluded MgATP from the enzyme-substrate complex
and furthermore, that the MgATP complex was the actual substrate (other
than glucose) for muscle hexokinase. Muscle hexokinase was also
activated by Cat", Co+,, Mn"+ and, to a very small extent by Zn'-. The
maximum activities for these activators were lover than that for Mg+
and the Km for each was lower than that for Mgt. The molar ratio
(metal/ATP) for maximum activation by the ions was less than 1. Strong
inhibition occurred as the concentrations of the metals were increased
above those giving maximum activation. They explained the differences
between these activators and Mg++ as being due to the different metal
ions combining with different ligand centers on the enzyme.
Walaas and Walaas (99) also reported that inosinetriphosphate (ITP),
guanidinetriphosphate (GTP) and uridinetriphosphate (UTP) would not
substitute for ATP in the muscle hexokinase reaction.
Griffiths (40) found that 0.005 alloxan inhibited muscle hexokinase
completely and 0.005M ninhydrin inhibited the enzyme 80 per cent.
Inhibition by 0.003M alloxan was reversed by 0.005M cysteine, but 0.01M
cysteine only partially reversed inhibition by 0.0025M ninhydrin. He
interpreted the results as indicating that the inhibition probably
amounted to more than just thiol destruction since reversal required a
molar ratio of cysteine to alloxan that was greater than 1.
Liver hexokinase. Long (63) measured levels of hexokinase in
several rat tissues and found that liver had the lowest level of hexokinase.
Crane and Sols (17) described a purification procedure for the
soluble hexokinase of rat liver. They concentrated a 100,000XG super-
natant fraction of liver extract with ammonium sulfate up to 50 per cent
saturation. Their liver preparation catalyzed phosphorylation of
glucose, mannose, fructose, glucosamine and 2DOG. There was some
evidence that fructose and glucose might have been phosphorylated by
separate enzymes. Lange and Kohn (58) found that allose, talose and
gulose were also substrates of rat liver hexokinase. They studied
hexokinase in rat intestine, kidney and liver extracts and their data
indicated that while the hexokinases from the three sources were similar,
they were not identical. The Km for glucose, 2DOG and glucosamine were
4X10-5, 9X10-5 and 3.7XI04M, respectively, and the relative maximal
velocities were in the same order.
Vinuela et al. (98) detected two enzymes in rat liver extracts
which catalyzed phosphorylation of glucose. One enzyme precipitated
between 20 and 50 per cent saturation with ammonium sulfate, while the
second enzyme precipitated between 60 and 70 per cent saturation. The
first enzyme had a low Km for glucose, was inhibited by G6P, and only
moderately by N-acetylglucosamine. The latter enzyme had a higher Km
for glucose, was not inhibited by G6P, was strongly inhibited by
N-acetylglucosamine, and had a low Km for mannose. They interpreted
the characteristics of the first enzyme as being those of a typical
animal hexokinase. It was relatively stable and was present in small
amounts in normal liver. They designated the second enzyme, a gluco-
kinase. It showed a maximal rate for glucose phosphorylation of
approximately 1.0 micramole per minute per gram liver at 21 to 230C or
2 micromoles at 37C which was comparable to the rate of glycogen
synthesis from glucose in rat liver (0.4 to 1 micromole per minute
per g liver). The activity also approached the maximum rate of
glycogen synthetase (3 micromoles per minute per g at 37C). The
glucokinase disappeared in starved and diabetic animals. They sug-
gested that the physiological instability of the glucokinase accounted
for the inability of liver to synthesize glycogen from glucose and
mannose in diabetic animals, while fructose and galactose, which were
incorporated into glycogen, were phosphorylated by other hexokinases
specific for those sugars. The Km for glucose of the hexokinase and
of the glucokinase were found to be X105M and 1X0I2M, respectively.
The glucokinase was not active on fructose to any extent. Their con-
clusion was that the glucokinase was associated with the glycogen
synthesizing system of the liver since: (1) its activity was compa-
rable with the rate of glycogen synthesis, (2) it was not inhibited
by its product and (3) UDPG-glycogen glucosyltransferase is dependent
on high levels of G6P for maximum activity.
Walker and Rao (100) examined the effects of 2DOG, glucosamine
and N-acetylglucosamine on the hexokinase and glucokinase of rat liver.
The three compounds inhibited both enzymes competitively, but the Ki
for 2DOG was much higher for the glucokinase than for the other two
compounds. The Ki for 2DOG was comparable to the In for glucose for
Kidney hexokinase. Kalckar (51) showed that kidney extracts
catalyzed phosphorylation of glucose and fructose. Colovick et al.
(14) demonstrated phosphorylation of mannose by kidney extracts.
Mannose isomerase in the extract converted M6P to F6P.
Lange and Kohn (58) found the Km for glucose, 2D00 and glucosamine
to be 4.8X10"5M, 9X1O'5M and 0.1M, respectively. Allose and talose were
also phosphorylated, while altrose and N-acetylglucosamine were not. It
appeared that kidney hexokinase differed from hexokinases of other rat
tissues (and other animal and plant hexokinases) in its specificity
towards modification at carbon atom 2 since glucosamine was such a
Intestine hexokinase. Sols (87) examined the hexokinase of rat
intestinal mucosa, and found fructose was phosphorylated at rates less
than glucose, while galactose, 3-methylglucose, L-sorbose, manno-
heptulose, N-acetylglucosamine, xylose, ribose and L-arabinose were
not phosphorylated. G6P at 6X10"fI inhibited hexokinase activity by
50 per cent, which was ten times the concentration required to inhibit
the brain enzyme to the same extent. All the sugars were phosphorylated
by the same enzyme. The Km for glucose and fructose were 2XIOT0 and
&X10-3M, respectively. These results failed to support the hypothesis
that the rate of phosphorylation of sugars limits the rate of sugar
absorption by intestinal mucosa since galactose and 3-methyl glucose,
which were not phosphorylated, were absorbed at faster rates than other
sugars (except glucose). Fructose, whose rate of absorption was inter-
mediate, was phosphorylated at a rate greater than any of the other
Lange and Kohn (58) found the Km for glucose, 2DOG and glucosamine
of rat intestine hexokinase to be 6.5X10o5, 9X10-5 and 3.3X101M,
respectively. Allose and talose, as with kidney hexoklnase, were also
active. Changes at carbon atom 3 of glucose (allose) had considerably
greater effect on activity than changes at carbon atom 2(2DOG or
glucosamine). Changes at either carbon atoms 2 and 3(altrose) or 3
and 4(gulose) resulted in loss of all activity. They concluded that
all the active substrates of kidney, liver and intestine hexokinase
were similar in at least two respects: (1) They had an available
hydroxyl at carbon atom 6 and (2) they had a hydroxyl available at the
anomeric position (carbon atom i).
Erythrocyte hexokinase. Christensen et al. (1l) found that the
hexokinase of rat erythrocyte phosphorylated glucose, mannose and
fructose at the relative rates 1.0:0.77:0.34 respectively. Galactose
was not active. The activity of normal or diabetic rats was not
affected by insulin or adrenal cortical extracts.
Retina hexokinase. Hoare and Kerly (44) showed that extracts of
rat retina phosphorylated glucose, fructose, mannose and glucosamine.
Magnesium ion, manganese ion and to a lesser extent, cobalt ion
activated dialyzed extracts. Glucose and glucosamine inhibited
phosphorylation of fructose.
Krebs 2 ascites tumor hexokinase. McComb and Yushok (69)
partially purified the hexokinase of Krebs-2 ascites tumor. The hexo-
kinase had a pH maximum between 5.6 and 7.8. The Km for glucose and
wtM vere 1.TX104 and 1X10'3M, respectively. The Ki for G6P inhibition
was 4XIO, while M6P, 2DOG6P and F6P were not inhibitory. Adenosine
diphosphate was inhibitory. Glucosone (Km 8X10'6M) was also phos-
phorylated by the tumor enzyme. The hexokinase activity was localized
in the mitochondria of both tumor and normal tissues.
Domestic fowl hexokinases. Mskata et al. (71) examined tissue
extracts from several organs of domestic fowl (white leghorn hen) and
found that the heart and gizzard hexokinases were active with both
glucose and fructose, while the enzyme of liver phosphorylated fructose
but not glucose.
Honey bee hexokinase. Ruiz-Amil (81) partially purified hexokinase
from honey bee by fractional precipitation of the enzyme between 50 and
70 per cent saturation with ammonium sulfate and column-chromatography
on DEAE-cellulose. The yield was 28 per cent of the original hexokinase
in the crude extract. The thorax contained the largest amount of hexo-
kinase activity (75 per cent). The enzyme showed optimum activity
between pH 7.5 and 8.8. Glacose-6-phosphate behaved as a competitive
inhibitor of glucose, while 2DOG6P and L-glycerophosphate did not
inhibit the enzyme to any extent. The KI for ATP and the Ki for its
competitive inhibitor, ADP, were 7.5X10'4M and SX10 M, respectively.
The Km for fructose (3X103SM) was similar to that of the mammalian
brain enzyme, while those for glucose (4X10"5M) and mannose (II0"-M)
were similar to those of yeast hexokinase. Ruiz-Anil concluded that
the enzyme from honey bee was similar to the hexokinase of other
animals in most respects.
Locust hexokinase. Kerly and Leaback (53) studied the hexokinase
in the thorax muscle and the salivary gland of locust (Locusta
migratoria). The properties of the enzyme were similar to those of
the n mmallan brain enzyme with respect to substrates and inhibitors.
Glucose had a Km of 5.5X10"3M. Both 06P and "6P, at a concentration
of 5i10'3M, completely inhibited hexokinase activity.
House fly hexokinase. Chefurka (10) observed hexokinase activity
in a soluble extract of house fly (Musca domestic L.).
Sea urchin egg hexokinase. Krahl et al. (56) studied hexokinase
in egg and embryo homogenates of sea urchin (Arbacia punctulata). The
enzyme was shown to have a pH optimum of 7. The substrate specificity
of the enzyme was similar to that of the other animal hexokinases. The
Km for the substrates that were examined fell in about the same range
(2 to 8X10-SM).
Bivalve hexokinase. Mekata et al. (71) observed that the
hexokinase of the fresh-water mussel (Hyrpsis schleeli) phosphorylated
fructose but not glucose.
Worm hexokinase. Bueding and MacKinnon (7) purified the hexokinase
of the parasitic worm, Schistosoma mansoni, 15- to 20-fold with the aid
of calcium phosphate and alumina Cy gels. They found that the worm
contained four distinct hexokinases: (1) glucokinase, (2) fructokinase,
(3) mannokinase and (4) glucosaminekinase. The glucokinase was
inhibited by ADP, G6P, sorbose-l-phosphate (SIP), and glucosamine-6-
phosphate. The inhibition of the glucokinase by ADP was noncompetitive
with respect to ATP. The Km for glucose, Mg++ and ATP were higher for
worm glucokinase than for those of mammalian brain hexokinase. Worm
fructokinase did not phosphorylate L-sorbose in contrast to fructo-
kinase of mammalian liver and muscle. Worm glucokinase was inhibited
by sulfhydryl inhibitors and such inhibition was reversed by glutathione.
Echinococcus hexokinases. Agosin and Aravena (1) partially
purified the hexokinases of hydrated cyst scolices of Echinococcus
granulosus. They isolated four separate hexokinases: (1) glucokinase,
(2) fructokinase, (3) mannokinase and (4) glucosaminekinase.
2-Deoxy-D-glucose was not phosphorylated by any of the hexokinases.
Gluco-, manno- and fructokinase were inhibited by G6P, while M6P
inhibited gluco- and mannokinase. Phosphorylation of fructose by
fructokinase was competitively inhibited by ADP. Glucokinase was
inhibited by p-chloromercuric benzoate (PCMB) and the inhibition was
reversed by cysteine.
Spirochaeta hexokinase. Smith (84) examined a hexokinase of the
particulate system of Spirochaeta recurrentis. The enzyme showed
maximum specificity toward glucose and mannose, while fructose and
L-sorbose were phosphorylated to a lesser extent. Galactose was not
active. Glueosamine and N-acetylglucosamine inhibited glucose phos-
phorylation. The enzyme was quite sensitive to low concentrations of
Pseudomonas hexokinases. Klein (55) observed that extracts of
Pseudomonas putrefaciens catalyzed phosphorylation of glucose and
glucosamine. The hexokinase of the extracts showed a very low affinity
for fructose (0 to 10 per cent relative to glucose) and mannose (0 to 5
per cent). Phosphorylation was not inhibited by 0.012M G6P. Sulfhydryl
(SH) poisons inhibited the hexokinase and the inhibition was reversed by
Hochster and Watson (45) reported that extracts of Pseudomonas
hydrophila had a distinct pentokinase (a xylokinase) that catalyzed
phosphorylation of xylose (Km = 2.3X103M).
Escherichia hexokinase. Cardini (8) partially purified the
hexokinases of Escherichia coll. Extracts of E. coli phosphorylated
glucose, fructose and mannose at relative rates of 1.00:0.14:0.29,
respectively. He observed that acetone or alcohol precipitation
removed the phosphorylating ability of the extracts for fructose,
while that for glucose and mannose remained constant, indicating that
a separate kinase was responsible for fructose phosphorylation in the
Staphylococcus hexokinase. Cardini (8) found the hexokinase of
Staphylococcus aurens was active only with glucose as the substrate.
He observed that normal cells of E. coli and S. aurens could be induced
to phosphorylate galactose in the carbon 1 position by growing the
cells on lactose.
Mycobacterium phlei inorganic polyphosphate glucokinase. Szymona
and Ostrowski (92) isolated an inorganic polyphosphate glucokinase
from Mycobacterium phlei which appeared to phosphorylate glucose by a
direct transfer of phosphate from inorganic polyphosphates containing
more than four phosphate residues. The apparent Km for inorganic poly-
phosphate and glucose were 1.75X0In M and 2.8XI0 lM, respectively.
Yeast hexokinase. Berger et al. (6) and Kunitz and MacDonald
(57) reported methods for crystallizing hexokinase of yeast at the
same time. Both methods were based mainly on fractional precipitation
of the enzyme with ammonium sulfate and ethanol from a toluene treated
extract. The enzyme was crystallized from an ammonium sulfate solution
and recrystallized up to six times.
Darrow and Colovick (23) reported a simpler method that gave a
good yield and high purity. Their method employed fractional precipi-
tation of the enzyme with ammonium sulfate, absorption and elution
from bentonite gel and repeated crystallization from ammonium sulfate.
The method gave about 150-fold increase in specific activity over the
The Km for substrates and Ki for inhibitors, as well as relative
maximum velocities (compared to glucose) reported by Darrow and
Colovick are given in Table 1. The specificity of the enzyme for
substrates and inhibitors, except for G6P, was similar to that of
brain hexokinase. However, the Km's were a magnitude smaller and
yeast hexokinase failed to phosphorylate mannoheptulose. Gottschalk
(39) presented evidence that indicated that the furanose ring con-
figuration of fructose might be the form which was active with yeast
The Km for ATP and Mg++ were found to be 10X10-5 (68) and
260X10-5M (6), respectively. The enzyme was specific for ATP. Deoxy-
ATP, ITP, UTP, CTP(cytosinetriphosphate), CTP, deoxy-CTP, deoxy-GTP
and adenosine tetraphosphate were ineffective as substitutes for ATF
(23) in the reaction. The requirement for Mg was not replaced or
antagonized by Ca+ (2).
Kaji at al. (50) showed that crystalline hexokinase, which was
prepared by the method of Darrow and Colowick (23), had a weak ATPase
activity which paralleled the hexokinase activity through six
crystallizations. Hexokinase inhibitors such as N-acetylglucosamine
and sorbose-l-phosphate inhibited the ATPase activity of the
hexokinase preparations. They observed parallel inactivation of the
ATPase and hexokinase activities by various amounts of silver nitrate.
Chromatography of the crystalline hexokinase on DEAE-cellulose gave
fractions in which the ATPase activity/hexokinase activity was
identical. The Em of the ATPase for ATP was 5X10'3M compared to
IX10'4M for that of the hexokinase. The hexokinase and ATPase were
found to have distinctly different specificity towards nucleotides.
Berger et al. (6) reported that thiol compounds provided no
protective action for their crystalline enzyme, while Bailey and Webb
(3) reported that all SH poisons, including Lewisite, were powerful
inhibitors of crystalline yeast hexokinase. Dixon and Needham (25)
reported inhibition of yeast hexokinase by mustard gas, while Stromme
(91) demonstrated that disulfiram was a potent inhibitor of the
enzyme, as was diethyldithiocarbamate when oxidized to disulfiram by
cyctochrome C. Barnard and Raael (4) obtained results that indicated
one to four -SH groups per molecule were required for the active
centers) of yeast hexokinase and that these groups ware not normally
available for reaction with -SH reagents, but probably became available
after a time-dependent structural change occurring at 300C. Berger
et al. (6) found that fluoride at 0.1254 did not inhibit crystalline
hexokinase when )Mg+ and phosphate were present in concentrations of
6.5 and X10-3M, respectively, but Bailey and Webb (3) observed that
0.04M NaF inhibited hexokinase activity 46 per cent.
Glucose-6-phosphate has been reported to be both inhibitory to
(101) and without effect (13, 102) on yeast hexokinase. From and
Zere (33) presented data that was interpreted as indicating that G6P
acts as a competitive inhibitor of both forward reaction substrates.
They suggested that the concentrations of A P used by other investi-
gators may have been too high to see inhibition. Trayser and Colowick
(96) reported the dissociation constant of G6P to be 4X10-3M, while
a ,nces and Kochavi (42) found it to be Xo10"'.
Strauss and Moat (89) reported that biotin stimulated fermentation
of glucose and fructose by air-dried yeast grown in a medium deficient
in the vitamin. Biotin also stimulated hexokinase activity in extracts
from cells grown on deficient medium. Trayser and Colovick (94) found
fully active yeast hexokinase did not contain biotin. The results
indicated that the enzyme was not a metalloprotein and did not contain
a readily demonstrable prosthetic group.
Crystalline yeast hexokinase has been reported to be a protein
of the albumin type with a manima molecular eight (MW) of 96,000
(3, 57) and a minimum MW of 30,000 (57). Its isoelectric point and
greatest stability is at pH 4.8 (3, 57). It has a pH optimum at
pH 7.5 (88). It has a turnover number (TN) of 13,000 moles per 105 g
protein per minute at 300C and pH 7.5 (6). The Q10 of the reaction
between 0 and 300C has been reported to be approximately 1.9 (6) and
is rapidly inactivated at temperatures above 55-600C (88).
Six-times crystallized hexokinase was separated into at least two
major components by either starch gel electrophoresis or DEAE-cellulose
column chromatography (23). The forms, A and B, obtained by column
chromatography were reported by Darrow and Colovick (22) to be
indistinguishable in specific activity, in Km values for substrates
and in sensitivity to inhibition by various substrates. However,
Trayser and Colowick (97) reported that the crystalline enzyme was
separated into six molecular forms (isozymes) of equal specific
activity by DEAE chromatography. The isozymes showed different
catalytic properties with respect to Km, Vmax, pH optima, ATPase
activity and sensitivity to inhibitors.
Berger et al. (6) found that the crystalline enzyme shoved a
loss of activity when highly dilute, which could be prevented by
diluting the enzyme in the presence of small amounts of insulin
(6 micrograms per ml) or serum albumin (60 micrograms per ml).
Glucose, fructose and to some extent mannose, prevented inactivation
of the enzyme in crude preparations and by trypsin. Insulin also
protected against inactivation by dilute alkali.
Several investigators (35, 52, 80), have demonstrated the
reversibility of the hexokinase reaction by measuring exchange of
either C14-labeled glucose between G6P and glucose or P32-labeled ADP
between ADP and ATP. However, the equilibrium greatly favored G6P
Agren and Engstrom (2) isolated phosphoserine from an acid
hydrolyzate of purified yeast hexokinase incubated in P32-labeled
ATP or G6P. They suggested that the hexokinase reaction involves the
formation of a stable phosphoenzyme intermediate. NaJJar and McCoy
(77) ruled out the phosphoenzyme hypothesis for yeast hexokinase when
they found no exchange of phosphorus between C1l-labeled glucose and
C2-labeled G6P (or vice versa) in the presence of the enzyme. They
then postulated the following mechanism for the reaction:
(1) Glucose-enzyme +ATP.-> 6-phosphoglucose-enzyme + ADP
(2) 6-Phosphoglucose-enzyme + glucose ->.glucose-enzyme + G6P
They argued that the formulation accounted for: (a) the lack of
exchange of phosphorous between glucose and G6P, (b) labeling of the
enzyme with G6P32 or ATP32 on the assumption that labeling of
phosphoserine might have been due to the transfer of phosphorous
during acid hydrolysis of the protein, (c) the marked inhibition of
the enzyme by G6P, and (d) C14-glucose labeled the enzyme. They con-
tended that when G6P accumulates, the greater part of the enzyme
would exist in the phosphoglucose-enzyme form, thereby bringing about
a corresponding reduction in the glucose-enzyme concentration which
would retard the forward rate of the first reaction and consequently
inhibit the overall reaction. They reported that C1-labeled glucose
labeled 30-60 per cent of the enzyme. One flaw in their formulation
is that there is no report in the literature that G6P markedly
inhibits yeast hexoklnase.
From and Zewe (33) examined the kinetics of the yeast hexokinase
reaction and they interpreted their results as being consistent with
the "classical" mechanism in which glucose and ATP add randomly to
the enzyme and equilibrium kinetics prevail. Their data indicated
that participation of either a phosphoenzyme complex or a glucoenzyme
complex was unlikely. Their results showed that mannose and ADP
behaved as competitive inhibitors of glucose and ATP, respectively.
Inhibition by AMP was competitive with respect to ATP and noncompeti-
tive with respect to glucose.
Trayeer and Colowick (95, 96) come to essentially the same
conclusions as Froma and Zewe (33) since they could not detect either
a phosphoenzyme complex or a glucoenzyme complex in their kinetic
studies of the yeast hexokinese reaction.
Hamess and Koehavl (41, 42, 43) did a detailed study of the
kinetics of the yeast hexokinase reaction. They derived from their
data what they considered to be the most probable mechanism for the
reaction. It involved the combination of MgATP and a glucoenzyme
complex to form two quaternary intermediates which in turn decomposed
to MgADP and a dissociable 6-phoaphoglucoenzyme complex. They pre-
sented the following compulsory pathway mechanism which is similar to
that postulated for brain bexokinase:
(1) Mg- + ATP-g-- ATP
(2) E + G- G complex
(3) E G + EMgAP X-- XA E 0 + GADP
(4) E 06P E + G6P
(5) MSADP -- .++ + ADP
here E, G, X1, X2 and G6P are enzyme, glucose, first quaternary com-
plex, second quaternary complex, and glucose-6-phosphate, respectively.
They did point out, however, that it is possible that both substrates
may have to be present at the same time in order for the enzyme to
have the correct conformation for phosphate transfer. Their data
revealed that g + may not be a very important factor for binding of
the substrate to the enzyme, but that the primary role of Mg++most
likely is to polarize the oxygen-phosphate bond that is being broken,
while anchoring the phosphate group of ATP to the enzyme. Nuclear
magnetic resonance studies indicated that binding of MgATP to the
enzyme might occur through the adenine and/or ribose portion of ATP.
Hales and Kochavi (42) observed a large difference in the
binding constants for glucose and GP by the enzyme and interpreted
it as indicating that either the hydroxyl group at carbon atom 6 of
glucose was important in the binding process or that the electrostatic
effect of the charged phosphate group inhibited binding of 06P to the
enzyme. They further concluded that G6P broke binding of 4gATP very
effectively. It can be seen from the foregoing that the mechanism of
the yeast hexokinase reaction is unclear, and Fromn and Zeve (33) point
out that the differences may be in the assumptions made and in the
interpretation of data.
Aspergllus hexokinase. Davidson (2e) purified the hexokinase of
Aspergillus parasiticu 225-fold. He employed fractional precipitation
with amonium sulfate (50-70%), followed by fractionation with cold
acetone (38-46%), negative absorption to alumina Cy gel, and refractiona-
tion vith ammonium sulfate (60-80%). The purified enzyme phosphorylated
D-glucose, D-galactose, D-glucosamine, D-galactosamine, D-mannose and
D-fructose at the relative rates (based on glucose) of 1.00:0.86:0.58:
0.40:0.67:0.36, respectively. L-sorbose and L-arabinose were active
to a very small extent. The enzyme phosphorylated galactose and
galactosamine to yield the respective 6-phosphates. The Em for glucose
and galactose were almost identical (l.6X10 M compared to 4.3XIOp- ).
They interpreted their data as indicating that all the substrates were
phosphorylated by the same enzyme and that the substrate specificity
suggested that the hexolinese was different from that of either the
yeast or brain enzyme.
Neurospora hexokinase. Medina and Nicholas (70) purified the
hexokinase of Neurospora crassa 60-fold. The enzyme phosphorylated
glucose and at a lower rate, mannose, fructose and glucosamine. The
enzyme was noncompetitively inhibited by G6P and was competitively
inhibited by N-acetylglucosamine. Iodoacetate, EDTA and PCMB were also
inhibitors of the enzyme.
Higher plants. Saltman (82) demonstrated the occurrence of
hexokinase in several plants and purified the soluble hexokinase of
wheat germ 5-fold. The hexokinases of the plants that were examined
were distributed between insoluble and soluble fractions. The distri-
bution depended on the tissue and the method of preparation of the
An insoluble hexokinase preparation was used for characterization
of the enzyme. Saltman's results, however, showed that the soluble and
insoluble enzymes were almost identical in their properties.
The enzyme phosphorylated glucose, fructose, mannose and
glucosamine, in the presence of ATP and Mg++, at the relative rates
1.00:0.62:0.68:0.52, respectively. The Km for glucose was 4.4X10'A
and that for ATP was 8.7X104M, while the Km for glucose by the soluble
enzyme was 4.6X10'4. Galactose, ribose, arabinose, ribulose, adenosine,
glyceraldehyde, dihydroxyacetone, mannitol and glucose-l-phosphate (GIP)
were inactive as substrates. Inosinetriphosphate (ITP) was 35 per cent
as effective as ATP. Magnesium ion activation was optimum at a concen-
tration of the ion equal to the concentration of ATP. Activation by
Mn"+ was 80 per cent as effective as Mg+ at 0.01M, while Co++ was
ineffective. Cupric, zinc and mercuric ions strongly inhibited the
enzyme. Potassium, sodium and ammonimu ions neither activated nor
inhibited the enzyme. Substances which influence -SH groups had little
effect on either the soluble or insoluble hexokinase. Zinc ion
inhibited the enzyme noncompetitively. Dinitrophenol (5X10"3M) inhibi-
ted phosphorylation 70 per cent, while G6P (5XIO'3M) only inhibited 17
per cent. The wheat germ hexokinase was found to be similar to yeast
Mekata et al. (71) demonstrated the occurrences of hexokinase
in a variety of higher plants.
Itoh (48) partially purified hexokinase from homogenates of
soybeans, Ozuki beans and mung bean by fractional precipitation of the
crude homogenates with ammonium sulfate. The hexokinase of soybean
precipitated at about 60 per cent saturation. The highest activity of
soybean hexokinase appeared three days after germination at 260C.
Itoh and Inouye (49) separated three different hexokinases of
soybean by fractional precipitation with ammonium sulfate. They
identified: (1) a glucokinase, specific for glucose and glucosamine,
(2) a fructokinase and (3) a galactokinase, specific for galactose and
Comparison of Animal and Plant Hexokinases
It appears that animal and plant hexokinases are similar in
substrate specificity and, in many cases, Km. The principal differences
appear to be the following:
(1) Hexose-6-phosphates produce a more marked inhibition of
animal than of plant hezokinases.
(2) ADP inhibition of plant hexokinases appears to be
competitive with respect to ATP, while in animals it
appears to be of a more complex nature.
(3) The hexokinase reaction catalyzed by brain hexokinase from
animals seems to follow a compulsory pathway mechanism
involving a stable phospho- or glucoenzyme complex(es).
Although there is disagreement, it appears that the enzyme
in plants might promote a random interaction of glucose and
ATP in the presence of Mg", without the formation of stable
phospho- or glucoenzyme complexes (33, 95).
Hexokinase and Sugar Uptake
The subject of sugar uptake by various animal tissues has been
thoroughly reviewed (18, 72) and will not be gone into in detail,
except to point out a few findings concerning the role of hexokinase
in sugar uptake.
Lundsgaard (65) first proposed that the active absorption of
sugars depended upon the sequential phosphorylation and dephosphoryla-
tion of the actively absorbed sugar. He later abandoned the hypothesis.
Drabkin (26) revived the hypothesis and proposed that the driving force
of active absorption involved the phosphorylation of the sugar outside
the cell by hexokinase and dephosphorylation inside the cell by glucose-
Sols (87) and Crane and Krane (22) have studied the specificity
of intestinal hexokinase and sugar absorption, respectively, and have
demonstrated the specificity of the enzyme to be contrary to that of
absorption. Dratz and Handler (27) have reported that the labeling of
the sugar-phosphate pool with p32 is inconsistent with the
phosphorylation-dephophohrylation hypothesis. Landau and Wilson
(61) concluded from their data that absorbed glucose does not pass
through the G6P pool of hamster intestine.
Crane (18) points out that, in the intestine, all actively
absorbed sugars have the common structure: -
Sugars that are not actively absorbed have a large (3-0-butyl-D-glucose)
or ionized substituent on some part of the structure or lack one of the
essential features of the glucose molecule (2-deoxy-D-glucose or
fructose). It also appears that the pyranose form is essential;
specifically the Cl chair form. SH also points out that the configura-
tion specificity at carbon atom 2 doesn't necessarily hold for other
animal tissues. He observed that the process of sugar absorption by
the intestine follows Michaells-Mfnten kinetics, requires the presence
of the free sugar, and doesn't depend on the gross metabolism of the
substrate since 3-0-mnthyl-D-glucose, which is absorbed at a great rate,
is not metabolized.
Crane et al. (19) and Crane (21) observed that sugar uptake by
the intestine depends upon the presence of Na+ ions and that the extent
of accumulation depends on the external concentration of Na+. They
proposed that glucose, on entering the brush border cells of the intes-
tine, combines with a carrier-Na+ complex. The complex moves through
the diffusion barrier to the interior of the cell where the Na+ portion
of the complex is extruded back through the diffusion barrier in the
reverse direction by an energy requiring process, which is inhibited
by strophanthidin, leaving the glucose "trapped" in the cytoplasm
(e.g. the so-called sodium "pump" involving the membrane ATPase system).
Cirillo (9) has shown that the yeast cell transports various
nonfermentable sugars across the cell membrane and that both nonfer-
mentable and fermentable sugars appear to share a common membrane
Morgan et al. (76) have demonstrated, from their studies of glucose
transport and phosphorylation in the perfused heart of normal and
diabetic rats, that glucose uptake is controlled by the combined opera-
tion of two sequential steps, membrane transport and intracellular
phosphorylation, and that in the steady state the net rate of membrane
transport and intracellular phosphorylation are equal. In the absence
of insulin, glucose uptake is limited by membrane transport. In the
presence of insulin, glucose uptake is accelerated and glucose phos-
phorylation is increased. Phosphorylation becomes increasingly limiting
as the external concentration of glucose is raised and provides the
major limitation to glucose uptake. They point out that the apparent
Km for glucose phosphorylation by hexokinase in diabetic heart muscle
is at least 7 times higher than in normal tissue. They found insulin
in vitro to have no large, immediate effect on glucose phosphorylation.
Randle (79) reported that the uptake of glucose and the
accumulation of D-xylose in isolated rat diaphragm are accelerated by
inorganic phosphate, G6P, GIP, F6P, FDP, AMP and ATP. He points out
that the effect is not marked and that perhaps it is the phosphate
group which is responsible for the acceleration. Anoxia and
2,4-dinitrophenol were also found to accelerate glucose uptake, but
such treatment also increased the rate of uptake of substances
(sorbitol) which are not ordinarily absorbed by diaphragm tissue. He
suggests that uptake might be regulated by phosphorylation and dephos-
phorylation of a membrane carrier.
Horecker et al. (46) observed that a mutant strain of E. coli
(Kn6), which utilized galactose much more rapidly than glucose, in
contrast to the wild type, has a galactokinase that has a much higher
affinity for ATP than the glucokinase of the cell. The galactokinase
is also inducible in the wild type cells. When ATP is limiting in
extracts of either strain, galactose is a strong inhibitor of glucose
phosphorylation. The inhibition also occurs in vivo in the mutant,
but doesn't in the wild type.
The findings of Humphreys and Garrard (47) with respect to
glucose uptake by corn scutellum slices are reviewed in the introduction
and discussion sections of this dissertation.
Glasziou (37, 38) has reported detailed studies of sugar uptake
and transformation in immature sugar cane internodal disks. His
results do not indicate a role for hexokinase in sugar uptake by that
MATERIALS AND MBBODS
Corn grains (Zea mays L., var. Funk's G-76) were soaked
twenty-four hours in running tap water and planted, scutellum-side up,
on four layers of moist filter paper on trays. The trays were covered
with aluminum foil and placed in the dark at 23C for five days. The
scutella were removed from the germinated grains and placed in ice-cold
glass distilled water until all the scutella were harvested. All sub-
sequent steps were carried out in the cold or in an ice-water bath.
Preparation of the Enzyme
Extraction. The scutella were weighed and then ground in a
chilled Waring Blendor for one and a half minutes in four volumes (v/v)
of ice-cold 0.005M ethylenediaminetetraacetate (EDTA), 0.005M magnesium
chloride (MgC12) and 0.01M potassium chloride (KCl), pH 7.0. The use
of either two volumes or six volumes of extracting solution decreased
recovery of the hexokinase. Cysteine (0.005M) or glutathione (0.01M)
did not increase recovery.
The homogenate was squeezed through two layers of cheesecloth
and the filtrate was centrifuged at OC and 2,000XG for one hour. The
supernatant fraction was filtered through glass wool to remove the
fatty layer and was then centrifuged at 0C and 32,00XG for one hour.
The supernatant fraction was filtered through glass wool and saved.
This fraction was designated "crude homogenate."
Ammonium sulfate fractionation. The crude homogenate was made
50 per cent saturated in ammonium sulfate ((lii)2SO04) by slow addition
of the salt to the magnetically stirred solution. The solution was
allowed to equilibrate, with stirring, for fifteen minutes after the
addition of the salt. The solution was centrifuged at 12,000XG for
twenty minutes at OOC. The precipitate was dissolved in a minimum
amount of 0.05M potassium phosphate buffer, pH 7.0, and was dialyzed
against several four liter changes of the same buffer for forty-eight
hours. This fraction was designated "F-l".
The 50 per cent saturated supernatant fraction from the above
centrifugation was made 75 per cent saturated in ammonium sulfate and
handled in the same manner as F-l. After dialysis the dissolved
protein fraction was designated "F-2" and the supernatant fraction,
"F-3". The F-2 fraction contained most of the recoverable activity
(Table 3) and was further purified by three successive treatments
with alumina Cr gel. The first two treatments absorbed substantial
amounts of the ATPase (Table 4) and the final treatment absorbed the
hexokinase which was used for this research. The following paragraph
describes the procedure that was used to purify the enzyme with
alumina Cy gel.
Absorption and elution from alumina Cy gel. The F-2 fraction
was treated with solid alumina Cy gel (7.4 per cent solids, Sigma
Chemical Company) as follows: (1) The F-2 fraction was treated with
0.0137g gel per ml (equivalent to 2.4 mg solids per ml). The gel was
dispersed by stirring with a ground-glass homogenizer and was allowed
to stand, with occasional stirring, for about thirty minutes. The
mixture was centrifuged at 0C and 12,000XG for twenty minutes. The
supernatant fraction was decanted and saved for further treatment.
The precipitate was dispersed and eluted for four hours with a volume
of 0.2M ammonium sulfate equal to one-third the volume of the F-2
fraction. The mixture was centrifuged at 00C and 12,00OXG for twenty
minutes. The supernatant fraction was decanted, adjusted to pH 7-7.5
with solid tris(hydroxymethyl)aminomethane base (tris base), portioned
among several plastic centrifuge tubes and frozen at -200C until
needed. This adjustment was necessary because the enzyme was inacti-
vated during storage at pH's below approximately 7. This eluate was
designated "A". The precipitate was eluted a second time with the
same volume of 0.4M ammonium sulfate and treated in the same manner
as the previous eluate. It was designated "A-l". (2) More gel
(0.063 g per ml, equivalent to 4.7 mg solids per ml) was added to the
supernatant fraction from the prior gel treatment. The mixture was
handled as before and the two eluates designated "B" and "B-l".
(3) To the supernatant fraction of the second gel treatment was added
0.0951 g per ml of gel (equivalent to 7.1 mg solids per ml) and the mix-
ture centrifuged and eluted as before. The two eluates were designated
"C" and "C-l". Fractions C and C-1 were the preparations used to
obtain the results reported in this paper. Both preparations had
identical Km for glucose, ATP and Mg"e and were considered to be the
same enzyme. They contained some adenosinetriphosphatase (ATPase) and
phosphoenolpyruvic phosphatase (PEPase) activity. Sodium molybdate
(Na2Mo207), 0.002M, completely inhibited the PEPase. Fractionation
of C and C-1 with solid ammonium sulfate, which resulted in 80 to
90 per cent recovery, did not remove the two interfering enzymes and
only succeeded in concentrating them along with the hexokinase.
Two methods were used for the assay of hexokinase activity and
for characterizing the enzyme.
Method 1. The rate of glucose-6-phosphate (G6P) formation at
2500 was measured by following nicotinamide adenine dinucleotide
phosphate (NADP) reduction in the presence of excess glucose-6-phosphate
dehydrogenase (G6PD) spectrophotometrically at 340 millimicrons. The
standard reaction cuvette contained the following: glucose, 20 micro-
moles; MgC12, 20 micromoles; Adenosine-5'-triphosphate, 20 micro-
moles; NADP, 1 micromole; Tris buffer, pH 8.0, 180 micromoles; G6PD,
1EU; hexokinase preparation and water to 3.2 or 3.3 ml. The blank
cuvette did not contain G6PD. This method was used to measure: (1)
the rate of phosphorylation of glucose and fructose (coupled to
phosphoglueoisomerase), (2) competitive inhibition by nons.abstrates
and glucose-l-phosphate (GIP), (3) nucleotide activation and inhibi-
tion, (4) metal activation, and (5) pH and temperature optima, which
were examined by running the hexokinase reaction at the various pH's
and temperatures in a total volume of 6 ml with twice the above
ingredients, except for HADP and 06PD. The reaction was stopped at
the end of 10 min by placing the reaction tube in a boiling water bath
for 2 min. A 3 ml aliquot was assayed for G6P with NADP and G6PD.
Method 2. The rate of adenosine-5'-diphosphate (ADP) production
at 250C by hexokinase was determined by measuring the oxidation of
reduced nicotinamide adenine dinucleotide (HADH) in the presence of
excess phosphoenolpyruvate (PEP), pyruvic kinase (PK) and lactic
dehydrogenase (LD) spectrophotometrically at 340 millimicrons. The
standard reaction cuvette contained the following: Tris buffer, pH
8.0, 180 micromoles; NADE, 0.282 micromoles; PEP, 10 micromoles;
Na2Mo207, 6 micromoles; MgC12, 20 micromoles; ATP, 10 micromoles;
PK, 1 EU; LD, 1 EU; hexokinase preparation and water to 3.2 or 3.3 ml.
The blank cuvette contained no NADH and a control was included, which
contained no sugar, to measure ATPase activity. This method was used
to measure: (1) the rate of phosphorylation of mannose, 2-deoxy-D-
glucose, L-glucose, L-mannose, N-acetyl-glucosamine, ribose, xylose,
and galactose, and (2) inhibition by nonsubstrates and the hexose-6-
phosphates, namely, G6P, 2-deoxy-D-glucose-6-phosphate (2DOG6P) and
mannose-6-phosphate (l6P). Mannose-6-phosphate inhibition could not
be measured by the G6PD reaction because it contained fructose-6-
phosphate (F6P) which was converted to G6P by phosphoglueoisomerase
in the scutellum hexokinase preparation.
Activity determinations by the two methods gave identical rates
for glucose after correcting for ATPase activity. With both methods
the observed rates were constant over at least sixteen minutes after
an initial one or two minute lag period.
Assay for phosphofructokinase, phosphoglucomutase and glucose-6-
phosphatase activities. Phosphofructokinase was assayed by coupling
the phosphorylation of F6P to NADH oxidation in the presence of excess
aldolase, triosephosphate isomerase (TPI), and C-glycerophosphate
dehydrogenase (c-GPD). Phosphoglucomutase activity was determined
by Method 1 above, using GIP as the substrate. Glucose-6-phosphatase
was measured by the same method after incubation of the hexokinase
preparation with a Imown amount of G6P for ten minutes.
Protein was estimated using the Folin-Ciocalteu reagent method
of Lowery et al. (64) as described by Layne (59). The colorimetric
readings were made on the Beclman DU Spectrophotometer at 720 milli-
microns. Crystalline bovine serum albumin was used as the standard.
Chemicals and Enzymes
All the chemicals and enzymes used in this investigation were of
the highest purity obtainable commercially. The enzymes were pur-
chased from the California Foundation for Biochemical Research. The
nucleotides, IADP, NADH, PEP, G6P, 1sP, F6F, 2DOG, and 2DOG6P were
obtained from Sigma Chemical Company. The other chemicals were
obtained either from the above firms, or from Nutritional Biochemicals
Company or Fisher Scientific Company.
The hexokinase in the crude homgenate could not be assayed
satisfactorily because of interfering enzyme activities.
Table 3 outlines the results of a typical purification of the
hexokinase from the scutellum. Although the amount of hexokinase in
the crude homogenate could not be estimated accurately, Table 3 shows
an "apparent" 30-fold increase in specific activity of the 50 to 75
per cent saturated amonium sulfate fraction (F-2) over the crude
homogenate. From Table 3 it can be seen that almost all of the hexo-
kinase activity was precipitated between 50 and 75 per cent saturation
with ammonium sulfate. The F-2 fraction contained the following
enzymes which might interfere with either one or the other assay:
glucose-6-phosphatase (G6Pase), phosphoenolpyruvic phosphatase
(PEPase), phosphoglucoisomerase (PGI), and adenosinetriphosphatase
(ATPase), but did not contain phosphoglucomutase (PGM), phosphofructo-
kinase (PFK), 6-phosphogluconic dehydrogenase and enzymes that destroy
reduced HAD or NADP. The G6Pase activity should not have interfered
seriously with the G6PD assay method because the excess G6PD in the
reaction mixture should act as a trap for the G6P produced by the
Treatment of the F-2 fraction with alumina Cy gel resulted in a
2.3-fold increase in specific activity of the C-l fraction over the
PURIFICATION OF HEOKIXNASE
activity Total % Recovery
Fraction (micromoles/min/mg activity from
protein) (units*) F-2 fraction
Crude homogenate 0.002 2.80 --
F-I (0-50%(Nm( )2804) 0.002 0.57 ---
F-2 (50-75%(NHB)gSo0 ) 0.060 12.60 ---
A (1st alumina C7 ) 0.026 0.64 5.1
A-l Igel treatment 0.012 0.11 0.9
B (2nd alumina ca-) 0.070 1.83 14.5
B-1 (gel treatment ) 0.049 0.81 6.4
C (3rd alumina C ) 0.102 3.45 27.4
C-1 (gel treatment ) 0.141 3.91 31.0
*One unit amount of enzyme that will phosphorylate 1 micromole of
glucose per minute at 250C.
F-2 fraction. This amounted to an "apparent" 70-fold increase over
the crude homogenate. When the F-2 fraction was dialyzed against the
extraction solution (EDTA-Mg-ECl) the APase activity paralleled the
absorption and the elution of the hexokinase activity from the gel,
but dialysis of the fraction against 0.05M potassium phosphate buffer,
pH 7.0, changed the absorption and elution characteristics of the pro-
tein with respect to the gel so that the ATPase activity could be
satisfactorily separated from the hexokinase activity as is shown in
Table 4. Buffers such as potassium phosphate, glycylglycine and tris
at several concentrations and several pH values were ineffective in
elating the hexokinase from the gel. Tris buffer in concentrations
above 0.1M inhibited the hexokinase activity irreversibly. The C and
C-l fractions, which were used as the enzyme preparations in these
investigations, contained PEPase activity which was completely
inhibited by 0.0012 sodium molybdate, a small amount of ATPase activ-
ity (Table 4) for which a correction was applied, and PGI activity.
The G6Pase activity of the F-2 fraction was not absorbed onto the gel.
The ATPase activity of the F-2 fraction was neither decreased by
centrifuging the crude homogenate fraction at 105,000XG for one and
one-quarter hours nor was it inhibited by ouabain (0.0005M to 0.005M)
or by fluoride (0.002M to 1.2M). The addition of Mg+ but not Na+or
K was required for ATPase activity. Since the content of ADP in the
reaction mixture was increased by the addition of AMP, part of the
"apparent ATPase" activity was due to adenylic kinase.
Further purification of the enzyme by acrylamide gel electro-
phoresis was attempted. The F-2 fraction separated into 11 or 12 bands
ATPASE ACTIVITY OF E ASE HEX E PEPAERATIICIS
activity Total % Recovery
Fraction (micromoles/min/mg activity from
protein) (units*) F-2 fraction
F-1 0.002 0.51 -
F-2 0.096 20.10
A 0.076 1.33 6.6
A-1 0.233 1.79 8.9
B 0.058 1.o4 5.2
B-1 0.134 1.55 7.7
C 0.025 0.58 2.9
C-i 0.020 0.38 1.9
*One unit amount of enzyme that will produce 1 micromole of
ADP per minute at 250C.
in the positive direction, while the C and C-i fractions separated
into four bands--two large bands between two smaller bands. The four
bands had Rf's corresponding to four similar bands separated from the
F-2 fraction. Hexokinase activity could be detected in one of the
bands, with Rf of 0.45-0.60, by slicing out the band, placing it in a
reaction cuvette and following the reduction of NADP at fifteen-
minute intervals up to one and one-half hours. The activity, 0.160
micromoles/cuvette/hour, was small and attempts to elute a significant
amount of the hexokinase from whole gel slices or homogenized slices
with several concentrations of glucose, phosphate buffer, ammonium
sulfate or glycylglycine buffer were unsuccessful. Most of the ATPase
activity in the gel was localized in a band with Rf of 0.77-1.0(calcu-
lated with reference to the distance traveled by the salt front). Such
a band had an activity of 0.037 micrcmoles/cuvette/hour. The other
bands also had some ATPase activity.
Acrylamide gel electrophoresis of commercial crystalline hexo-
kinase (CalBiochem) also gave four bands that had a separation pattern
similar to that of the C and C-1 fractions with the two large bands
having Rf's almost identical to those of the C or C-i fractions. Again
the recovery was small. Approximately 28 units of activity were added
to each gel and at the end of the run the gel was cut into small pieces
and eluted with 1 ml of 0.04M glycylglycine buffer, pH 7.5, for thirty
minutes. The resulting eluate had an activity of 0.224 units/mi or
about 0.9 per cent recovery.
Attempts to purify the enzyme by absorption and elution from
bentonite by the method described by Darrow and Colowick (23) were
unsuccessful. The hexokinase and ATase activities were absorbed
but could not be eluted.
Fractionation of the enzyme preparations at any step in the
outlined procedure with cold acetone or ethanol (-70C) resulted in a
complete loss of activity.
Humphreys and Garrard (47) found that slices of the corn scutellum
take up glucose at the rate of 70-80 micromoles/g fresh weight/hour
(approximately 1 unit per g of tissue) but do not accumulate it.
Therefore, if it is assumed that all the glucose entering the slices
is phosphorylated, the hexokinase content of the tissue should be much
higher than that recovered. Since the F-l and F-2 fractions would
account for only about one-fifth of the total apparent hexokinase
activity of scutellum, attempts were made to increase the yield.
Reduced glutathione or cysteine in concentrations of 0.005M and 0.01M
in the extracting solution did not increase the yield of hexokinase.
Including glucose in concentrations of either 1 or 10 per cent in the
extracting solution or in the crude homogenate also did not increase
the yield. Detergents such as deoxycholate (0.026 per cent) and
Triton X-100 (0.1 per cent) not only failed to increase the yield, but
also increased the solubility of lipid in the crude homogenates so that
it could not be removed by centrifugation followed by filtering through
glass wool. The lipid interfered with protein precipitation in the
subsequent salt fractionations by causing the protein to float to the
top of the centrifuge tubes with the lipid. The floating material was
difficult to collect quantitatively and the hexokinase activity in
this material was lower than that obtained by the method described in
the materials and methods section. Dialysis of the floating layer
did not solubilize the enzyme and treatment with cold acetone or
ethanol at -70C to remove the lipid destroyed the hexokinase activity.
Itoh (48) was able to use the floating layer to study the hexokinase
of soybean. Dimethylsulfoxide (DMSO) at concentrations of 0.1 and 1.0
per cent in the extracting solution did not affect the yield. Extracts
of acetone powders prepared from scutella yielded smaller amounts of
The endosperm of the geminated corn grains did not contain
detectable hexokinase activity.
Sugars. The specificity of scutellum hexokinase for sixteen
sugars was examined. The Michaelis constants (Io), maximum velocities
(Vmax) and relative Vmax with respect to glucose are given in Table 5.
The enzyme phosphorylated D-glucose, D-mannose, D-fructose, 2-deoxy-D-
glucose and D-glucosamine. Glucose had the lowest Km (6.X10"'5S) and
the greatest Vmax. The substrate concentration versus rate curve for
glucose and the corresponding Linewever-Burk plot (62) are presented
in Figures 1 and 2, respectively.
Nucleoside triphosphates. The Km for ATP was found to be 8X10'M.
Figures 3 and 4 show the curves from which this value was calculated.
Commercial preparations of UTP (uridinetriphosphate), GTP(guanosine-
triphosphate), CTP (cytidinetriphosphate) and TTP (thymidinetriphos-
phate) were found to be poor substitutes for ATP at the concentrations
that were examined (Table 6). The best substitute for ATP was TP which
gave 28 per cent of the rate of 0.0015M ATP at a concentration of 0.003M.
SUBSTRATE SPECIFICITY OF CORN SCUTELLUM HEXOINASE
Substrate (MX105) Vmax"* Vax
D-glucose 6.4 0.047 1.00
D-mannose 9.3 0.028 0.59
D-fructose 168 0.046 0.97
2-deoxy-D-glucose 30 0.044 0.94
D-glucosamine 37 0.030 0.63
*Inhibition constant (Ki)
*O.D./2 min/cuvette at 250C
"No detectable activity or inhibition at 0.03M
The low rates obtained by these compounds could have been due to a
small amount of nucleoside diphosphokinase activity for which the
preparations were not assayed. Saltman (82) observed that inosine-
triphosphate (ITP) was 35 per cent as effective as ATP as a phosphate
donor in the hexokinase reaction catalyzed by an insoluble hexokinase
from wheat. Walas and Walaas (99) could not detect hexokinase
activity with ITP, GTP or UTP with their preparations from muscle
Divalent metal cations were required for scutellum hexokinase
activity. Table 7 gives the NI and a comparison of the activation of
scutellum hexokinase by M C+, Co+ and Mn-. Figures 5, 6 and 7 show
the metal concentration versus rate curves and the Lineweaver-Burk
plots for the three metal cations. Magnesium ion was by far the best
activator. An inhibition of magnesium activation was observed when
the molar ratio of ATP/Mg exceeded approximately four. When the ratio
was 7.5 micromoles/1 micronole or 7.5/2 micromoles the rate, compared
to 2 micromoles/l micromole or 2 micromoles/2 micromoles, was 20 and
10 per cent lower, respectively, and caused the LIneweaver-Burk plot
for MgS to deviate from linearity. Therefore, the ratio was always
maintained less than four.
Manganese ion was inhibitory at concentrations greater than the
concentration of ATP (0.003M) (Table 8 and Figure 5) which indicates a
maximal activation by the ion when the molar ratio ATP/Mn is one. The
inhibition did not occur when such a ratio was maintained. Such
inhibition is similar to that observed by Walaas and Walaas (99).
r) ( J-
Ol x u1'WF Ja9d '70V
Figure 2. Lineweaver-Burk plot of the
effect of glucose concentration on the rate
0 2 4 6 8 10 12 14 16
EFFECT OF NUCLEOSIDEIPHOSPHATFS
AS SUBSRTIRTES FOR AP
Concentration % of Rate Obtained
Nucleosidetriphosphate (M3103) vith 1.510"-3M
UTP 1.5 19
GTP 1.5 14
CIP 1.5 14
TIP 1.5 15
*Mg+ concentration constant at 6X10'3M
Figure 3. Effect of ATP concentration
on the rate of phosphorylation, and competitive
inhibition by ADP and AMP. Reaction cuvettes
contained: 180 micramoles Tris buffer, pH 8.0;
20 micromoles glucose; MC12 in amounts equal
to, or greater than, the concentration of
inhibitor plus ATP; 1 micromole of NADP; 1 EU
G6PD; 10 miromoles of ADP or 20 micromoles of
AM; 0.1 ml enzyme preparation and water to
0 2 4 6
ATP Mx I03
ACTIVATION OF HEXOKINASE BY M!AL IONS*
Kn Vmax % VMax
Ion (Mno05) ( O.D./2 min/cuvette) Relative
M914 20 0.040 100
Co+" 70 0.025 63
Mn++ 100 0.020** 50**
%Ratio of ATP/metal ion was always maintained at less than 4.
"Calculated frcn extrapolated Lineveaver-Burk plot. Actual
observed relative VWas was 38 per cent.
Figure 5. Effects of Co+- and Mn"
concentrations on the rate of phosphorylation.
The standard reaction mixture was used, except
the concentration of ATP was 10 micromoles
Figure 6. Effect of Mg++ concentration
on the rate of phosphorylation. The standard
reaction mixture was used except: for the
first two concentrations of Mg 2.0 micro-
moles ATP were used and for the last four
concentrations, 7.5 micromoles ATP were
Mg++ MX 103
Figure 7. Lineveaver-Burk plots of the
effect of Mg~, Co++ and Mh++ concentration
on the rate of phosphorylation.
No inhibition by Mg++ and Co++ was seen at the concentrations employed
in these experiments.
The addition of potassium ion or sodium ion did not cause activa-
tion of the enzyme.
Sugars. Of the nonsubstrates listed in Table 5, N-acetylglucosa-
mine and D-xylose were found to be competitive inhibitors of glucose
with Ki of 55X10'5M and 87fOXO-M5, respectively. Figure 8 shows the
Lineveaver-Burk plots from which the Ki in Table 8 were calculated.
The other nonsubstrates had no inhibitory activity.
Nucleoside di- and triphosphates. ADP and AMP were found to be
competitive inhibitors of ATP with Ki of 14X10"5M and IXO'-3, respec-
tively. Figures 3 and 4 show the curves for inhibition by 0.003M ADP
and O.006M AMP. UTP, GI, IP, CTP, UDP and GDP were found to have
little inhibitory activity with respect to ATP at the concentrations
that were examined (Table 8). The slight inhibition of hexokinase by
these six compounds was not increased by increasing the concentration
of the nucleotides.
Sugar phosphates. No inhibition of glucose phosphorylation was
observed with G6P, F6P, MSP, GIP, galactose-6-phosphate (Gal6P) or
ribose-5-phosphate (R5P) at concentrations of the sugar phosphate up to
0.033M. The concentration of glucose in these experiments was that
which resulted in about half maimal velocity (6X10'SM).
Anions. Fluoride in concentrations of 0.002 to 1.2M did not
inhibit scutellum hexokinase. MgCl2 and gSO4 were equally effective
in activating the enzyme.
l ~I aI
0 0 0 0 0
(-)- in re N -
EFFECT OF N~CLEOSIDE DI- AMD TRIPHOSPHATES AS INHIBITORS
OF ATP IN THE HEXOKIASE REACTION*
% of Rate
Nucleotide Concentration** Relative to
(MX0o3) 1.5Xo-3M ATP Alone
UTP -- -
GTP 1.5 91
lTP ---- --
UDP 1.5 80
GDP 1.5 90
*M+ concentration constant at 6Xfl03M
c*ATP concentration vas 1.5X103M
pH and Temperature Optima
A pH optimum (Figure 9) was observed at pH 8.0, while the optimum
temperature for scutellum hexokinase was found to be 490C (Figure 10).
This optimum is similar to that observed for wheat germ hexo-
kinase (82), muscle hexokinase (99) and honey bee hexokinase (81).
The scutellum hexokinase lost activity rapidly when stored at pH's
The temperature optimum (Figure 10) is much higher than that
reported for other hexokinases. Hexokinase isolated by Saltman (99)
from wheat germ had a temperature optimum of 3700 but Taha and
El-Towesy (93) reported that phytases from several seeds including bar-
ley and wheat, had temperature optima in the vicinity of 51C. Berger
et al. (6) found that the Q10 for yeast hexokinase was close to 2 over
the range of 0-300C. The Q10 of the hexokinase from the corn scutellum
(Figure 10) is 2 between 20 and 300C but falls off at temperatures above
and below this range. The Q10 might vary with the enzyme, the com-
ponents of the reaction mixture and at higher temperatures can be a
reflection of the rate of denaturation and the rate of the reaction.
0 o oo r-- o uo re) N -
9/oI lnwL!XOk/ jO /Ua3J9c
u! WO/d-9-9 sOaloA
The results indicate that the pattern of substrate specificity of
scutellum hexokinase is similar to that of yeast hexokinase. The Km
of the scutellum enzyme for D-glucose, D-cannose, 2-deoxy-D-glucose
and ATP are of the same order of magnitude as those of the yeast
enzyme (Table 1). The Km for D-fructose is an order of magnitude
larger. However, Gottschalk (39) has shown that the furanose ring
configuration of fructose is the true hexokinase substrate and, at
equilibrium, about 20 per cent of a fructose solution is in the furanose
form. If this is the case with the scutellum enzyme, the true Ke would
be about five times smaller than the value given in Table 5. The Km
for glucosamine is an order of magnitude smaller than that of yeast
The most significant difference between the hexokinase frcm the
scutellum and that from brain or yeast is the relative rate of phos-
phorylation of the other four substrates caopared to glucose. Glucose
is phosphorylated at a rate greater than any of the other substrates.
Apparently the scutellum enzyme shows a greater specificity with
respect to differences at carbon atom 2 of the glucose molecule than
does the yeast or brain hexokinases.
Sols and Crane (86) suggest that the coefficient of phosphorylation
(. Vmax substrate X Km glucose ) is a true measure of the physiological
Vmax glucose Ka substrate
suitability of a substrate for hexokinase. With the scutellum enzyme
the coefficients of phosphorylation for D-glucose, D-mannose,
2-deoxy-D-glucose, D-glucosamine and D-fructose are 1.00:0.41:0.30:
0.11:0.04, respectively. These values indicate that D-glucose is the
most suitable physiological substrate for the scutellum hexokinase.
Correcting for the furanose configuration of fructose would place
fructose closer to (about the same as 2-deoxy-D-glucose), but still
lower than, glucose in suitability as a substrate.
The results from the limited studies of the substrate specificity
and of inhibition by the various nonsubstrates are essentially in
agreement with the findings of Sols et al. (88) for yeast hexokinase
and of Sole and Crane (86) for brain hexokinase. The scutellum hexo-
kinase is specific for the D-configuration since the L- forms of the
substrates are neither phosphorylated nor are they inhibitory. The
five-carbon sugars (ribose and xylose) are not phosphorylated but xylose
is a competitive inhibitor. The enzyme also shows specificity for
hydroxyls at carbon atoms 1(G1P and %-methyl-D-glucoside) and
3(3-0-methyl-D-glucose) and for inversion of the hydroxyl group at
carbon atom 4 since galactose and galactosamine were not active as sub-
strates or inhibitors. Inversion of the hydroxyl at carbon atom 2
might account for the low rate of phosphorylation of mannose and the
inversion at carbon atom 3 would account for ribose being noninhibitory.
No analogs of glucose which lack hydroxyla at carbon atom 6 were tested
except for the hexose-6-phosphates and the fact that they were not
inhibitory suggests that scutellum hexokinase requires a byaroxyl at
that position for binding. This is supported by the fact that xylose
is a weak competitive inhibitor.
Activation of the scutellum hexokinase by Mg+, Co++ and Mn++
closely resembles that observed by Walaas and Walaas (99) for muscle
hexokinase, although inhibition by MgH and Co+f was not observed at
the concentrations used in the experiments. Saltman (82) observed that
MnDb was 80 per cent as effective as MgH for activating the insoluble
hexokinase of wheat germ while Co++ was completely ineffective. The Km
(2X10 M) for activation of the seutellum hexokinase by M$g is a magni-
tude smaller than that reported by Walsas and Walaas (99) for the
muscle enzyme and by Berger et al. (6) for the yeast enzyme.
The results show that ADP and AMP appear to act as competitive
inhibitors of ATP. The Km for ATP and the Ki for ADP suggests that the
enzyme shows similar affinity for both nucleotides. Similar inhibition
has been demonstrated for yeast hexokinase (33) and honey bee hexokinase
(81). Inhibition by ADP with respect to ATP for brain hexokinase has
been reported to be noncompetitive (27) or of a complex nature (32).
In the experiments reported in this investigation the magnesium ion
concentration was maintained at levels equal to, or greater than, the
concentration of substrate (ATP) plus inhibitor (ADP or AMP) so that
there would be no inhibition caused by competition for magnesium ion.
Inhibition of the scutellum hexokinase reaction by G6P or by other
hexose-6-phosphates, as suggested by Humphreys and Garrard (47) from
their studies of glucose-uptake by scutellum slices, was not observed
in these investigations. The levels of AlT used in these experiments,
0.003 and 0.006M, may have mashed inhibition, as has been suggested by
Prom and Zeve (33) for yeast hexokinase, but such inhibition must not
have been very large. It may be that there is another glucose-
phosphorylating enzyme in the scutellum but the results indicate that
there are none.
When one considers that the role of the scutellum in the
germinating seedling is essentially that of a "sucrose factory" and
that it is capable of absorbing and utilizing glucose at a rapid rate
(1 micromole/min/g fresh weight at 300C) it seems reasonable that the
sucrose and energy synthesizing systems of the scutellum should be
efficient ones and may be analogous to the glycogen synthesizing sys-
tem of rat liver studied by Vinuela at al. (98). They demonstrated
that there was a glucokinase in the liver which was distinguishable
from the typical animal bexokinase of that organ. The glucokinase had
a higher IN for glucose, was not inhibited by G6P and phosphorylated
glucose at a rate which was comparable to the rate of glycogen synthesis
in the liver. Leloir et al. (60) reported that uridine diphosphoglucose-
glycogenglucosyltransferase was activated by high levels of G6P. The
system might also be similar to starch synthesizing system in seeds
which appear to be highly specific towards substrates (30). Also, the
hexokinase of yeast, an organism which utilizes sugars as its principal
source of energy, is not inhibited to any extent by its products (13,
33, 83, 102).
Humphreys and Garrard (47) observed that preincubation of scutellum
slices in water prior to introduction of glucose into the bathing
medium, increased the rate of glucose uptake by the slices. There was
a concomitant decrease in G6P in the slices with the length of preincuba-
tion which they interpreted as indicating that G6P was acting as a com-
petitive inhibitor of glucose phosphorylation and thereby limiting
glucose-uptake. They observed similar inhibition with preincubation in
mannose solutions, which could be partially reversed by washing the
mannose from the slices. The residual inhibition was attributed to
the high level of MSP. The results reported in this paper suggest
that G6P and MSP are not inhibitors of scutellum hexokinase, and the
factors controlling glucose uptake in the scutellum must be other than
inhibition of the hexokinase reaction by hexose-6-phosphates. M6P has
not been found to be an inhibitor of brain (16) or yeast hexokinase
(83), but might well be inhibiting some step in the utilization of G6P.
2-Deoxy-D-glucose has been shown to be a competitive inhibitor of G6P
for the phosphoglucoisomerase of rat kidney (103). Both the availabil-
ity of ATP at the site of the hexokinase reaction and ADP inhibition
could affect the rate of glucose phosphorylation and consequently the
rate of glucose uptake. Preincubation might cause an increase in
available AIP and removal of inhibitory ADP. At the same time, ATP
availability and nucleosidediphosphate inhibition could be affecting
the utilization of G6P by limiting the rate of uridinediphosphoglucose
pyrophosphorylase through the nucleoside diphosphokinase reaction, and
preincubation would reflect increases in high-energy phosphate compounds
at these sites with an accompanying decrease in G6P. Various investi-
gators (30, 34) have demonstrated strong interrelationships between
nucleotides and between nucleotide-sugar compounds in starch and sucrose
synthesis. Morgan et al. (76) have shown that glucose-uptake in the
heart of rat is controlled by both transport and intracellular phos-
phorylatlon. When membrane transport is not limiting (i.e. sufficient
insulin is available) the rate of phosphorylation limits the rate of
glucose-uptake at high levels of exogenous glucose.
It is believed that the results presented in this paper support
the conclusion that the hezokinase of the corn scutellum is more
correctly a glucokinase which is not inhibited by its product, G6P.
A hexokLnase was extracted from the scutellum of corn and
purified with an "apparent" 70-fold increase in specific activity.
It was free of interfering enzyme activities and appeared to be the
only soluble hexokinase of the scutellum. The enzyme showed greatest
specificity towards glucose as the substrate and the data supports
the conclusion that the hexokinase is more specifically a glucokinase.
The enzyme phosphorylated D-glucose, D-mannose, D-fructose, D-glucos-
amine and 2-deoxy-D-glucose. It was specific for ATP as the phosphate
donor. N-acetylglucosamine and xylose were competitive inhibitors of
glucose phosphorylation, while ADP and AMP vere competitive inhibitors
of ATP. The divalent cations of magnesium, cobalt and manganese
activated the enzyme with cobalt and manganese ion being 63 and
38 per cent as effective as magnesium, respectively. The hexokinase
was not inhibited by hexose-6-phosphates at concentrations up to
500-times the glucose concentration. Optimum activity of the enzyme
was observed at pH 8.0 and 490C.
The properties of the hexoklnase from the scutellum are compared
with those of the brain and yeast hexokinases and are discussed in
relation to glucose-uptake by the scutellum.
1. Agosin, Moises and Luisa Aravena. Studies on the metabolism of
Echinococcus granulosus. II. Glycolysis with special references
to hexokinases and related glycolytic enzymes. Biochim. Biophys.
Acta 34: 90. 1959.
2. Agren, G. and L. Engstrcm. Isolation of 32-labeled phosphoserine
from yeast hexokinase preparation, incubated with labeled ATP or
glucose-6-phosphate. Acta. Chem. Scand. 10: 489. 1956.
3. Bailey, K and E. C. Webb. Purification of yeast hexokinase and
its reaction with f('-dichlorodiethyl sulphide. Biochem. J.
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Herbert Charles Jones III was born October 6, 1936, in
Leesburg, Lake County, Florida. He attended public schools in
Ocala, Florida and was graduated from Ocala High School in June,
He attended the Colorado School of Mines and the University of
Florida before entering the United States Air Force in 1956. While
in the service he married the former Winifred Catherine Gassaway of
Jacksonville, Florida. After two years of active duty with the Air
Force he returned to the University of Florida and was graduated
August, 1960, with the Degree of Bachelor of Science in Forestry.
In September, 1960, he began graduate study at the University of
Florida, majoring in Botany. In June, 1965, the Degree of Doctor of
Philosophy was conferred on him.
He is the father of three children: Catherine Anne, Winifred
Gail and Herbert Charles.
He is a member of the following honorary societies: Xi Sigma PI,
Gamma Sigma Delta, Alpha Zeta and Phi Signa.
This dissertation was prepared under the direction of the
chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to
the Dean of the College of Agriculture and to the Graduate Council,
and was approved as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
June 22, 1965
--7 SW ^-^
Dean, College of Agriculture
Dean, Graduate School