30 Review TRENDS in Biochemical Sciences Vol.26 No.
1 January 2001
PFK-2/FBPase-2: maker and breaker
of the essential biofactor fructose-2,
6-bisphosphate
David A. Okar, Ànna Manzano,Aurèa Navarro-Sabatè, Lluìs Riera, Ramon Bartrons
and Alex J. Lange
Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated
gluconeogenesis in the liver.This discovery has led to the realization that this
compound plays a significant role in directing carbohydrate fluxes in all
eukaryotes. Biophysical studies of the enzyme that both synthesizes and
degrades this biofactor have yielded insight into its molecular enzymology.
Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential
in the treatment of diabetes.
A trail that began with the discovery of a highly potent
regulator of mammalian hepatic carbohydrate
metabolism, fructose-2,6-bisphosphate (F-2,6-P2), led
to the discovery of the bifunctional enzyme
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase (PFK-2/FBPase-2) that is
responsible for both the formation and degradation of
this compound, and to the genes that code for it. Since
the discovery of this system in liver, other mammalian
tissue-specific bifunctional isozymes and their genes
have been identified. F-2,6-P2 and the enzymes
responsible for controlling the amount of this biofactor
appear to be present in all eukaryotes, including
plants and yeast. Although the particular function of
F-2,6-P2 in each cell type varies to some extent,
adaptation to changing environmental or metabolic
situations is a common theme and this implies that a
diversity of mechanisms have evolved to control the
relative kinase (synthetic) and bisphosphatase
(degradative) activities. The most striking example of
this diversity is the yeast, which do not express a
bifunctional enzyme, rather they use a set of enzymes
David A. Okar
Alex J. Lange* that have one or the other activity diminished by
Dept of Biochemistry, ‘mutation’ of key catalytic amino acid residues in the
Molecular Biology and kinase or bisphosphatase active sites. This highlights
Biophysics, University of
Minnesota, Minneapolis,
the complexity of this convoluted signaling enzyme
MN 55455, USA. system, which is an essential determinant in the
*e-mail: regulation of carbohydrate metabolism.
lange024@tc.umn.edu
Ànna Manzano A multimodulated bifunctional enzyme
Aurea Navarro-Sabatè F-2,6-P2 was discovered during the search for the
Lluìs Riera
Ramon Bartrons
mechanism by which glucagon stimulates hepatic
Unitat de Bioquímica, gluconeogenesis. The fact that it does not participate as
Campus de Bellvitge, an intermediary in any metabolic interconversion, as
Universitat de Barcelona,
well as its lability in acid extracts used in systematic
C/Feixa Llarga, S/N, 08907
L’Hospitalet, Barcelona, surveys of phosphoric acid esters in tissues, explains
Spain. why it escaped discovery until 1980. Eventually,
http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01699-6
extraction in base led to the discovery of F-2,6-P2 and
the realization that it was either formed or destroyed
during metabolic transitions where its concentration
determined changes in glycolytic and gluconeogenic
carbon flux in the liver. Interest in this compound grew
rapidly because it was found to be a most potent
positive allosteric effector of 6-phosphofructo-1-
kinase (PFK-1; EC 2.7.1.11) and an inhibitor of
fructose-1,6-bisphosphatase (FBPase-1; EC 3.1.3.11).
Because of its antagonistic actions on these enzymes,
F-2,6-P2 plays a crucial role in the control of the
opposing hepatic glycolytic and gluconeogenic
pathways1–4 (Fig. 1). Because glycolysis requires the
presence of F-2,6-P2, it is important in all glycolysis-
dependent tissues and it has since been found in
virtually every eukaryotic tissue or cell examined5.
Whether it is a fungus switching to an alternative
carbon source, a turtle hatchling freezing in a nest or a
germinating seed, F-2,6-P2 is intimately involved in the
metabolic fine tuning required for survival.
A single enzyme family is responsible for
determining the levels of F-2,6-P2 by synthesizing and
degrading this compound at distinct active sites. That
is, the kinase (EC 2.7.1.105) synthesizes F-2,6-P2 from
ATP and fructose-6-phosphate (F-6-P), whereas the
bisphosphatase (EC 3.1.3.46) degrades F-2,6-P2 to
F-6-P and inorganic phosphate (Pi). It should be
appreciated not only that the enzyme catalyzes
reciprocal reactions, but also that it does so as a dimer
that is stabilized by numerous protein–protein
interactions between the kinase domains (Fig. 2)6. By
contrast, the crystal structure of the rat testis
bifunctional enzyme suggests that the bisphosphatase
domains have little, if any, contact across the dimeric
interface. This view is supported by the observation
that the separately expressed rat liver bisphosphatase
domain is monomeric, even at greater than 4 mM
(80 mg ml−1)7. The quaternary structure of the rat
bifunctional enzyme is relevant to the monofunctional
yeast isozymes because they retain the two domain
structure of the monomers and are dimeric8,9.
However, the ‘monofunctionality’ refers to the subunits
of the dimer. Considering that the expression of a
single yeast isozyme is not exclusive, it is possible that
the yeast enzymes demonstrate bifunctionality by
 Review TRENDS in Biochemical Sciences Vol.26 No.1 January 2001 31

(cAMP)-dependent protein kinase cascade set off by

Glucose glucagon binding to the extracellular site of its receptor.
This compound forms the juncture between the
hormonal signal pathway and the metabolic pathway.
GK Glu-6-Pase Because both the Km of PFK-2 and the Ki of FBPase-2
for F-6-P are in the physiological range, any alteration
of F-6-P concentration should cause inverse changes in
the two activities. The reciprocal modulation of the
Glucose 6-P
kinase and bisphosphatase activities forms the current
paradigm for explaining how the bifunctional enzyme
Fructose 6-P can closely regulate the level of F-2,6-P2 in vivo.
The kinase reaction proceeds by a sequential-
PFK-2/FBPase-2 ordered mechanism for transfer of the phosphate from
ATP to F-6-P. The bisphosphatase reaction proceeds via
KK
Citrate PKA K K a covalent phosphohistidine intermediate formed upon
Gly-3-P
Gly-3-P_ reaction with F-2,6-P2. In isolation, neither reaction is
PEP
B B B B + Pi
freely reversible. The steady state concentration of
PP2A
PFK-1 FBPase-1 F-2,6-P2 is determined by the balance between these
opposing reactions. This kinase:bisphosphatase activity
Fructose 2,6-P2
ratio (K:B) is the most salient aspect of any given
_
+ bifunctional enzyme isoform, whether the active sites
reside on the same monomeric component or not,
because the balance between the reciprocal reactions is
Fructose 1,6-P2 what determines the net effect on the cellular content of
+ F-2,6-P2. The K:B is determined by which isoforms are
present, the levels of several glycolytic and
PEP
gluconeogenic metabolites, post-translational
PEPCK modification of the enzyme, and even xylulose-5-
phosphate, an intermediate of the hexose
PK OAA monophosphate pathway. Metabolites beyond
PFK-1, such as α-glycerol phosphate,
phosphoenolpyruvate and citrate decrease the activity
Pyruvate
of PFK-2 or favor that of FBPase-2, in accordance with
the concept of a negative feed-back control loop11,12. In
addition, the bisphosphatase activity is modulated by
Lactate, alanine Ti BS GTP and ATP, which activate FBPase-2 at
subsaturating substrate concentrations, but inhibit
Fig. 1.The position of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) in competitively at saturating substrate concentrations13.
the pathways of hepatic intermediary carbohydrate metabolism. Regulation of PFK-2/FBPase-2 The vast range of signals impinging upon the
target activities, 6-phosphofructo-1-kinase (PFK-1) and fructose-1,6-bisphosphatase (FBPase-1), as
well as regulation of PFK-2/FBPase-2 itself via effectors and covalent modification are shown.
bifunctional enzyme constitute the enzyme’s
Abbreviations: Gly-3-P, glyceraldehyde-3-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; multimodality and, together, determine the overall K:B
PEPCK, PEP carboxykinase; Pi, inorganic phosphate; PK, pyruvate kinase; PKA, protein kinase A; and thereby the F-2,6-P2 content of the living
PP2A, protein phosphatase 2A.
cell1–5,11,12,14,15. The significance of F-2,6-P2 with regard
forming mixed dimers in which the monomers possess to carbohydrate flux has been exploited to lower the
reciprocal kinase and bisphosphatase activities. blood glucose levels in streptozotocin-treated mice,
Several years ago, Sols et al. described the concept of which model type I diabetes. This was done using
enzyme multimodulation arising from the adenovirus-mediated overexpression of the rat liver
accumulation of several regulatory mechanisms in a bifunctional enzyme engineered to have a high K:B
given enzyme10. These regulatory mechanisms can be of (Ref. 16). The engineered enzyme has two mutations,
different types (cooperative, allosteric or Ser32Ala and His258Ala, which remove a regulatory
interconversion), of the same type (multiple allosteric phosphorylation site and a key component of the
effects) or of any combination. PFK-2/FBPase-2 has bisphosphatase active site, respectively.
many of these properties, making it responsive to a Overexpression of the high K:B bifunctional enzyme
plethora of metabolic and hormonal signals. This is markedly stimulated glucokinase (GK) expression and
consistent with its central role in regulation of diminished that of glucose-6-phosphatase (G-6-Pase).
carbohydrate metabolic fluxes in a diverse cross-section This indicates that the cellular effects of F-2,6-P2 are
of tissue types and eukaryotic life-forms. In the liver, multimodal, effecting the appropriate reciprocal
F-2,6-P2 is derived directly from the fructose- regulation of GK and G-6-Pase, as well as metabolic
6-phosphate/glucose-6-phosphate (F-6-P/G-6-P) pool flux, by reciprocal modulation of PFK-1 and FBPase-1
and is the ultimate messenger in the cyclic AMP activities.

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32 Review TRENDS in Biochemical Sciences Vol.26 No.1 January 2001

atase
A B
us Bisphosph
in Kinase C terminus [F-2,6-P2 ]
rm
te
N K >1

N
B

te
rm
Kinase C terminus HGP

in
Bisphos

us
phatase

Protein kinase A Protein phosphatase 2A

s
inu Kinase
P m Bisphosp
er hatase
[F-2,6-P2 ] N t
C te
rmin
K us
<1
B i nus
N rm
te C te
HGP P rm osphatase
inu Kinase Bisph
s
Ti BS

Ti BS
Fig. 2. A trace of the back-bone structure of the rat testis bifunctional
enzyme (2BIF)6.The dimer interface is shown by a solid black line and
the monomers are designated as A and B. Kinase domains are in red
and green; bisphosphatase domains in blue and magenta.
The liver isoform of the bifunctional enzyme
switches between a high K:B state (>1) and a low K:B
state (<1), depending upon the phosphorylation state
of Ser32 (Fig. 3)17. A decrease in the K:B is due to both
a reduction of the kinase activity and an
enhancement of the bisphosphatase activity upon
phosphorylation of Ser32 in response to glucagon18.
By contrast, upregulation of the F-2,6-P2 levels is
responsive to glucose via stimulation of a xylulose-5-
phosphate-dependent protein phosphatase that
preferentially dephosphorylates Ser32 in the hepatic
bifunctional enzyme19,20. Although the mechanism
remains unknown, the effect of Ser32
phosphorylation on the K:B appears to be mediated
by the N- and C-termini of the enzyme because
deletion of the N-terminal 22 amino acids and
truncation of the C-terminal 30 amino acids produced
a bifunctional enzyme that was still phosphorylated,
but without effect on the activities21.
Bisphosphatase in detail
Rarely has it been possible to obtain an X-ray
structure and high-field nuclear magnetic resonance
(NMR) spectra from an enzyme during turn-over of
the physiologically relevant substrate, yet the
extreme stability of the covalent phosphohistidine
intermediate of the bisphosphatase has allowed the
acquisition of structural and spectral data22–24. The
stable intermediate is phosphorylated at the 3′-N of
His258 and is complexed with F-6-P; the
dissociation of which appears to be the rate-limiting
step in the bisphosphatase reaction7,25. As such, the
Fig. 3. Regulation of the liver bifunctional enzyme activities by post-
translational modification. Phosphorylation of Ser32 (indicated by the
yellow circle) activates the bisphosphatase and inhibits the kinase.
Dephosphorylation of the same residue produces the opposite effects.
This inversion of the kinase:bisphosphatase (K:B) ratio modulates the
hepatic content of fructose-2,6-bisphosphate (F-2,6-P2) as indicated,
which subsequently effects the hepatic glucose production (HGP).
Glucagon stimulates the cyclic AMP (cAMP)-dependent protein kinase
A, and glucose stimulates the xylulose-5-phosphate-dependent protein
phosphatase 2A.Therefore, the opposing metabolic effects of glucose
and glucagon can be accounted for, at least in part, by their respective
effects on the Ser32 phosphorylation state of 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase.
transition state analogs provided many insights into
the mechanisms of biocatalysis. The bisphosphatase
and the serine proteases both use histidine as a
crucial component of their respective catalytic
machinery, however, they employ a different
constellation of residues at the active site to exploit
the amphipathic nature of the imidazole ring to
drive different reactions. The 15N-, 13C- and 1H-NMR
spectral signatures of the histidine(s) in these
enzyme families allows a comparison of the histidine
at work in two different reaction mechanisms.
The NMR spectroscopic analyses of the serine
proteases26,27 have revealed strong hydrogen bonds
involving the imidazole nitrogens, designated low-
barrier hydrogen bonds (LBHBs)28. Although the
precise role of LBHBs in catalysis remains
controversial, it is clear that they are significantly
stronger than the average hydrogen bond. The
transition state for hydrolysis of a peptide bond would
probably be a higher energy state than the stable
intermediate for the hydrolysis of an acid-labile sugar
phosphate. Consistent with this view, the
bisphosphatase does not form LBHBs, as judged by
the criteria of the chemical shifts for nuclei within the
histidine ring during turnover23. Conversely, the 15N,
13C and 1H chemical shifts of His258, phosphorylated

bisphosphatase during turnover is similar to the or not, and the adjunct His392, correspond well with
serine proteases, where the detailed structural and another phosphohistidine-containing
spectral studies of the enzymes in complex with phosphotransferase, IIIGlc of Escherichia coli (Ref. 29).

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Review TRENDS in Biochemical Sciences Vol.26 No.1 January 2001 33

Table 1. Bifunctional isozyme propertiesa

PFK-2 FBPase-2
µΜ)
Km (µ µΜ)
Km (µ
Isozymes Amino acids Mr Vmax F-6-P ATP Vmax F-2,6-P2 Kinase/ Refs
(mU/mg) mU/mg phosphatase
Rat liver 470 55 113 35 150 45 <0.1 2.5 12
Bovine liver 470 55 42 150 12 35 7 1.2 45
Fetal rat liver ND 55 80 44 195 ND ND ND 46
Rat muscle 450 54 66 56 48 154 0.4 0.4 47
Bovine heart 570 58 61 74 0.26 33 40 1.8 48
Rat testis 468 55 90 85 270 22 21 4.1 30
Human testis 468 55 75 58 650 80 16 0.9 49
Bovine brain ND 120 90 27 55 29 70 3.1 50
Human placenta 520 59 142 32 220 0.2 130 710 49
aAbbreviations:F-2,6-P2, fructose-2,6-bisphosphate; F-6-P, fructose-6-phosphate; FBPase-2, fructose-2,6-bisphosphatase; ND, none detected or
not determined; PFK-2, 6-phosphofructo-2-kinase.

Different needs, different isozymes
Since the discovery of rat liver PFK-2/FBPase-2,
many more mammalian isozymes have been
identified in skeletal muscle, heart and brain, as
well as a ubiquitous isozyme that is present in
placenta and tumor cells. The testis30 and the
liver12 isozymes are the most similar (75%
identity), followed by liver and heart31 (73%
identity). The ubiquitous, or placental,
PFK-2/FBPase-2 shows the greatest divergence
from the liver isozyme. Although the core structure
of the different PFK-2/FBPase-2 enzymes is highly
conserved, there are differences in kinase and
bisphosphatase properties among the different
isozymes (Table 1), presumably, due to the
structural variations in the terminal regions.
The bifunctional enzyme monomer can be
thought of as being composed of four regions, with
the core kinase and bisphosphatase domains at the
center, and regulatory regions at the termini. These
observations are borne out by comparison of the
PFK-2/FBPase-2 amino acid sequences, which show
high identity in the core catalytic regions.
Alignments of the bifunctional enzyme isozymes
can be accessed at the Protein Information
Resource (PIR; http://pir.georgetown.edu/pirwww/).
Given that the bifunctional enzyme functions as a
dimer, the terminal regions can elicit their
regulatory function by changes in the tertiary or
quaternary conformations in response to the
multiplicity of effectors. The localization of the
dimerization site within the kinase domain and the
proximity of the Ser32 phosphorylation site favors a
change in quaternary structure as the general
scheme by which the K:B might be inverted.
The gene expression of PFK-2/FBP-2 is regulated
by hormones and metabolites12, yet the expression of
tissue-specific isoforms of PFK-2/FBPase-2 in their
respective tissues is not completely exclusive. In
liver, for example, 10% of the expression is from the
skeletal muscle isoform32. Also, within tissues that
are made up of different cell types, different isoforms
of the bifunctional enzyme are expressed. The
placental/ubiquitous isoform, and not the liver
isoform, is expressed only in the Kupffer cells of the
liver33. The expression of more than one isozyme in
the same tissue suggests that different isozymes play
key roles in different physiological conditions or in
response to specific hormones.
At least six different isoforms of the bifunctional
enzyme have been identified in mammals and all are
generated by alternative splicing of the transcribed
RNA from only four genes, designated PFKFB1–4
(Table 2)34. Consistent with the divergence of the

Table 2. Properties of bifunctional isozyme genesa

Localization
Gene Size (Kb) Human Rat Isozymes mRNA (Kb)
Liver 2.1
PFKFB1 (A) 60 (rat/human) Xq27–q28 Xq22–q31 Muscle 1.9
Fetal 2.2

Heart 4/6.8
PFKFB2 (B) 22 (rat/human) 1q31 13q24–q25 Kidney 4/6.8
Pancreatic islets

Placenta 5.4
PFKFB3 14 (human)b 10p15–p14 Ubiquitous 4.8
Inducible

PFKFB4 40 (human)b 3p22–p21 Testis 2.4

aAbbreviation:
PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
bDeduced from GenBank data and Refs 51, 52.

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34 Review TRENDS in Biochemical Sciences Vol.26 No.1 January 2001
amino acid sequences in the terminal regions, it is
the use of alternative exons in processing the
primary transcript to generate various mRNAs that
give rise to the numerous isoforms30,35–39. For
example, the rat liver, muscle and fetal isozymes are
transcribed from the same gene, but the liver protein
contains a consensus phosphorylation site for cAMP-
dependent protein kinase (Ser32)12, whereas the
others do not36. This is consistent with physiological
demand, because when the liver is exporting glucose
and the peripheral tissues are using it, the F-2,6-P2
level in the liver must be relatively low to minimize
glycolysis, and that in the peripheral tissue must be
high to promote it. This balance can be maintained
only if the relevant bifunctional enzymes are
differentially regulated.
Evolutionary design of a bifunctional enzyme
It appears that the use of F-2,6-P2 as a regulatory
metabolite is a specifically eukaryotic
phenomenon. The most plausible hypothesis for the
origin of the PFK-2/FBPase-2 would be the fusion
of two ancestral genes coding for a kinase
functional unit and a phosphohydrolase/mutase
unit, respectively14. From protein sequence
alignments, it is clear that the bisphosphatase
activity located in the C-terminal domain of the
PFK-2/FBPase-2 is homologous to the
phosphoglycerate mutases (PGMs) and the acid
phosphatase family40. Alignments of the
bisphosphatase domain with PGM and acid
phosphatase can be accessed at the PIR website
(http://pir.georgetown.edu/cgi-bin/pirwww/
nbrfget?uid=DA3761&db=A). The N-terminal
PFK-2 domain is sequentially and structurally
homologous to several nucleotide binding proteins,
primarily that of adenylate kinase of E. coli
(Refs 6,41; Fig. 4).
Clearly, the emergence of a discrete nucleus in the
early eukaryotes must have required the
development of new metabolic control mechanisms to
augment those of the prokaryotic cells from which it
had so recently evolved. Apparently, F-2,6-P2 was one
biofactor that could fill this role. Interestingly,
NMPK Phosphohydrolase/
mutase
PFK-1
Fig. 4. Convergent and
divergent aspects of
PFK-2/FBPase-2 evolution PFK-2
and gene fusion.
Abbreviations: FBPase-2, Mutase
fructose-
2,6-bisphosphatase;
NMPK, nucleoside
Gene fusion
monophosphate kinase; Acid phosphatase
PFK-1, 6-phosphofructo-1-
kinase; PFK-2, PFK-2/FBPase-2
6-phosphofructo-2- Ti BS
kinase.
http://tibs.trends.com
although E. coli do not express any PFK-2 activity,
and therefore do not contain any F-2,6-P2, their
PFK-1 is still activated by F-2,6-P2 via the same
allosteric mechanism demonstrated in the
mammalian enzymes.
Among the eukaryotes, plants, yeast and animals
appear to have adapted the use of F-2,6-P2 in different
ways. Plants use F-2,6-P2 to partition carbohydrate
stores between sucrose and starch, which is somewhat
analogous to the role of the compound in mammals
(i.e. to direct fuel use and storage)16. Modulation of the
bifunctional enzyme K:B is similar in plants, fish,
reptiles, amphibia and mammals5. Yeast PFK-1 is
activated by F-2,6-P2 (Ref. 42), however as mentioned
earlier, the yeast have developed a different means to
control cellular F-2,6-P2 content.
Conclusion and prospects
We have attempted to show that since its discovery in
the early 1980s, the study of PFK-2/FBPase-2 has led
to a significant expansion in the number of genes and
isozymic gene products, as well as an increase in
structural and regulatory (both acute and long-term)
information about this enzyme. The detailed research
into the F-2,6-P2 system by a variety of methods and
in several tissues and organisms has illuminated a
complex set of interwoven control mechanisms that
make the cellular F-2,6-P2 content responsive to
many signals derived from the pathways of fuel
metabolism. Elucidating the role of F-2,6-P2 in the
regulation of hepatic fuel usage in the gilthead sea
bream, a commercial fish important to the economies
of the Mediterranean region, is one such example43.
Fundamental questions that have yet to be answered
include: What are the specific roles of the many
isoforms and how are different isozymes regulated
both acutely and by changes in gene expression?
Furthermore, how do the two domains, kinase and
bisphosphatase, communicate with each other to
affect the reciprocal regulation?
The importance of F-2,6-P2 to the regulation of
carbohydrate metabolism has made the bifunctional
enzyme a target for therapeutic intervention in
diabetes. Liver-targeted genetic delivery systems
using chimeraplasty44 are being tested in the
development of blood glucose-lowering therapies
using diabetic mouse models16. In heart, cardiac
expression-specific bifunctional enzyme transgenes
are under development in an attempt to raise
glycolytic rates to prevent diabetes-associated
cardiomyopathy (Q. Liang and P.N. Epstein,
unpublished). These efforts, as well as the in-depth
studies of the structure and mechanism of the
bisphosphatase domain, provide insight into the
function of this enzyme at the cellular and molecular
level23,24. It is hoped that the study of the bifunctional
enzymes and their genes will benefit those afflicted
with diabetes, while we gain a deeper understanding
of how organisms coordinately regulate the metabolic
pathways of life.
 Review
References
1 Hers, H. et al. (1982) Fructose-2,6-bisphosphate.
Trends Biochem. Sci. 7, 329–331
2 Hers, H. and Van Schaftingen, E. (1982) Fructose
2,6-bisphosphate 2 years after its discovery.
Biochem. J. 206, 1–12
3 Pilkis, S.J. et al. (1983) Rat hepatic
6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase: a unique bifunctional
enzyme. Adv. Enz. Regul. 21, 147–173
4 Uyeda, K. et al. (1982) Fructose-2,6-P2, chemistry
and biological function. Mol. Cell. Biochem. 48,
97–120
5 Okar, D.A. and Lange, A.J. (1999) Fructose-
2,6-bisphosphate and control of carbohydrate
metabolism in eukaryotes. Biofactors 10, 1–14
6 Hasemann, C.A. et al. (1996) The crystal
structure of the bifunctional enzyme
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase reveals distinct domain
homologies. Structure 4, 1017–1029
7 Okar, D.A. et al. (1995) Identification of transient
intermediates in the bisphosphatase reaction of
rat liver 6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase by 31P-NMR spectroscopy.
Biochem. J. 308, 189–195
8 Kretschmer, M. et al. (1987) Fructose-
2,6-bisphosphatase and 6-phosphofructo-2-kinase
are separable in yeast. Biochem. J. 246, 755–759
9 Boles, E. et al. (1996) Cloning of a second gene
encoding 6-phosphofructo-2-kinase in yeast, and
characterization of mutant strains without
fructose-2,6-bisphosphate. Mol. Microbiol. 20,
65–76
10 Sols, A. (1981) Multimodulation of enzyme
activity. Curr. Top. Cell. Regul. 19, 77–101
11 Van Schaftingen, E. (1987) Fructose
2,6-bisphosphate. Adv. Enzymol. Relat. Areas
Mol. Biol. 59, 315–395
12 Pilkis, S.J. et al. (1995) 6-Phosphofructo-2-
kinase/fructose-2,6-bisphosphatase: a metabolic
signaling enzyme. Annu. Rev. Biochem. 64,
799–835
13 Lee, Y.H. et al. (1994) Mechanism of modulation of
rat liver fructose-2,6-bisphosphatase by
nucleoside triphosphates. J. Biol. Chem. 269,
11002–11010
14 Pilkis, S., ed. (1990) Fructose 2,6-bisphosphate,
CRC Press
15 Kurland, I.J. and Pilkis, S.J. (1995) Covalent
control of 6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase: insights into autoregulation
of a bifunctional enzyme. Protein Sci. 4,
1023–1037
16 Wu, C. et al. Overexpression of 6-phosphofructo-
2-kinase/fructose-2,6-bisphosphatase in mouse
liver lowers blood glucose by suppression of
hepatic glucose production. J. Clin. Invest.
(in press)
17 El-Maghrabi, M.R. and Pilkis, S.J. (1984) Rat
liver 6-phosphofructo 2-kinase/fructose
2,6-bisphosphatase: a review of relationships
between the two activities of the enzyme. J. Cell.
Biochem. 26, 1–17
18 Pilkis, S.J. et al. (1988) Hormonal regulation of
hepatic gluconeogenesis and glycolysis. Annu.
Rev. Biochem. 57, 755–783
19 Nishimura, M. et al. (1994) Glucose-stimulated
synthesis of fructose 2,6-bisphosphate in rat liver.
Dephosphorylation of fructose 6-phosphate, 2-
kinase:fructose 2,6-bisphosphatase and activation
by a sugar phosphate. J. Biol. Chem. 269,
26100–26106
http://tibs.trends.com
TRENDS in Biochemical Sciences Vol.26 No.1 January 2001
20 Nishimura, M. and Uyeda, K. (1995) Purification
and characterization of a novel xylulose-5-
phosphate-activated protein phosphatase
catalyzing dephosphorylation of fructose-
6-phosphate,2-kinase:fructose-2,6-bisphosphatase.
J. Biol. Chem. 270, 26341–26346
21 Kurland, I.J. et al. (1992) Rat liver
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase. Properties of phospho- and
dephospho- forms and of two mutants in which
Ser32 has been changed by site-directed
mutagenesis. J. Biol. Chem. 267, 4416–4423
22 Lee, Y-H. et al. (1997) Crystal structure of a
trapped phosphoenzyme during a catalytic
reaction. Nat. Struct. Biol. 4, 615–618
23 Okar, D.A. et al. (2000) The mechanism of the
bisphosphatase reaction of 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase probed by 1H -
15N NMR spectroscopy. Biochemistry 39,
9754–9762
24 Okar, D.A. et al. (1999) The roles of Glu-327 and
His-446 in the bisphosphatase reaction of rat liver
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase probed by NMR spectroscopic
and mutational analyses of the enzyme in the
transient phosphohistidine intermediate
complex. Biochemistry 38, 4471–4479
25 Stewart, H.B. et al. (1985) Evidence for a
phosphoenzyme intermediate in the reaction
pathway of rat hepatic fructose-2,6-bisphosphatase.
J. Biol. Chem. 260, 12935–12941
26 Markley, J.L. and Westler, W.M. (1996)
Protonation-state dependence of hydrogen bond
strengths and exchange rates in a serine protease
catalytic triad: bovine chymotrypsinogen A.
Biochemistry 35, 11092–11097
27 Markley, J.L. (1978) Hydrogen bonds in serine
proteinases and their complexes with protein
proteinase. Biochemistry 17, 4648–4656
28 Cleland, W. and Kreevoy, M. (1994) Low-barrier
hydrogen bonds and enzymic catalysis. Science
264, 1887–1890
29 Pelton, J.G. et al. (1993) Tautomeric states of the
active-site histidines of phosphorylated and
unphosphorylated IIIGlc, a signal-transducing
protein from Escherichia coli, using
two-dimensional heteronuclear NMR techniques.
Protein Sci. 2, 543–558
30 Sakata, J. et al. (1991) Molecular cloning of the
DNA and expression and characterization of rat
testes fructose-6-phosphate, 2-kinase:fructose-
2,6-bisphosphatase. J. Biol. Chem. 266,
15764–15770
31 Sakata, J. and Uyeda, K. (1990) Bovine heart
fructose-6-phosphate,2-kinase:fructose
2,6-bisphosphatase: complete amino acid
sequence and localization of phosphorylation
sites. Proc. Natl. Acad. Sci. U. S. A. 87, 4951–4955
32 Colosia, A.D. et al. (1988) Induction of rat liver
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase mRNA by refeeding and
insulin. J. Biol. Chem. 263, 18669–18677
33 Sakakibara, R. et al. (1999) Tissue distribution of
placenta-type 6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase. Biochem. Biophys. Res.
Commun. 257, 177–181
34 Kumar, S. et al. (1994) MEGA: Molecular
evolutionary genetics analysis software for
microcomputers. Comput. Appl. Biosci. 12, 231–238
35 Darville, M.I. et al. (1987) Complete nucleotide
sequence coding for rat liver 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase derived from
a cDNA clone. FEBS Lett. 224, 317–321
35
36 Darville, M.I. et al. (1989) 5′-Flanking sequence
and structure of a gene encoding rat
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase. Proc. Natl. Acad. Sci. U. S. A.
86, 6543–6547
37 Rider, M. et al. (1992) The two forms of bovine
heart 6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase result from alternative
splicing. Biochem. J. 285, 405–411
38 Watanabe, F. and Furuya, E. (1999) Tissue-
specific alternative splicing of rat brain
fructose-6 phosphate 2-kinase/fructose
2,6-bisphosphatase. FEBS Lett. 458, 1–5
39 Ventura, F. et al. (1995) Cloning and expression of
a catalytic core bovine brain 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase. Biochem.
Biophys. Res. Commun. 209, 1140–1148
40 Bazan, J.F. et al. (1989) Evolution of a
bifunctional enzyme: 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase. Proc. Natl.
Acad. Sci. U. S. A. 86, 9642–9646
41 Bertrand, L. et al. (1997) Modelling the 2-kinase
domain of 6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase on adenylate kinase.
Biochem. J. 321, 615–621
42 Bartrons, R. et al. (1982) The stimulation of yeast
phosphofructokinase by fructose
2,6-bisphosphate. FEBS Lett. 143, 137–140
43 Meton, I. et al. (2000) 6-Phosphofructo-2-
kinase/fructose-2,6-bisphosphatase
gene expression is regulated by diet composition
and ration size in liver of gilthead sea
bream, Sparus aurata. Biochim. Biophys. Acta
1491, 1–3
44 Kren, B.T. et al. (1999) Correction of the UDP-
glucuronosyltransferase gene defect in the
Gunn rat model of Crigler–Najjar syndrome
type I with a chimeric oligonucleotide. Proc.
Natl. Acad. Sci. U. S. A. 96, 10349–10354
45 Kountz, P.D. et al. (1985) Isolation and
characterization of 6-phosphofructo-2-
kinase/fructose-2,6-bisphosphatase from bovine
liver. Arch. Biochem. Biophys. 238, 531–543
46 Martin-Sanz, P. et al. (1992) Characterization of
6-phosphofructo 2-kinase from fetal-rat liver.
Biochem. J. 281, 457–463
47 Kitamura, K. et al. (1989) Purification and
characterization of rat skeletal muscle fructose-
6-phosphate, 2-kinase:fructose-
2,6-bisphosphatase. J. Biol. Chem. 264,
9799–9806
48 Kitamura, K. and Uyeda, K. (1987) The
mechanism of activation of heart fructose-
6-phosphate, 2-kinase:fructose-
2,6-bisphosphatase. J. Biol. Chem.
262, 679–681
49 Sakakibara, R. et al. (1997) Characterization
of a human placental fructose-6-phosphate,2-
kinase/fructose-2,6-bisphosphatase. J. Biochem.
(Tokyo) 122, 122–128
50 Ventura, F. et al. (1992) Bovine brain
6-phosphofructo-2-kinase/fructose-
2,6-bisphosphatase. Evidence for a neural-specific
isozyme. J. Biol. Chem. 267, 17939–17943
51 Manzano, A. et al. (1999) Cloning, expression
and chromosomal localization of a human
testis 6-phosphofructo-2-kinase/fructose
2,6-bisphosphatase gene. Gene 229, 83–89
52 Manzano, A. et al. (1998) Molecular cloning
expression and chromosomal localization of a
ubiquitously expressed human 6-phosphofructo-
2-kinase/fructose 2,6-bisphosphatase gene
(PFKB3). Cytogenet. Cell Genet. 83, 214–217