Phosphoglucose Isomerase
Briana Betz, '03
Lisa Maurer, '04
Contents:
I. Introduction
Phosphoglucose isomerase, as its name suggests,
is the enzyme crucial for the interconversion of D-glucose 6-phosphate
and D-fructose 6-phosphate. PGI is the enzyme responsible for the
second step of glycolysis and is involved in glucogenesis (2).
Thus, it is highly conserved in bacteria and eukaryotes (2).
Besides functioning as an isomerase, PGI serves as a neuroleukin, autocrine
motility factor, and a differentiation and maturation mediator (2).
Neuroleukins, potent cytokins, are secreted by T-cells and promote the
survival of specific embryonic and sensory nerves (4).
In addition, neuroleukins allow B cells to mature into antibody secreting
cells (2). Autocrine motility factor
causes cancer cells to migrate and appears to be involved in tumor metasis
and invasion. Further, it has been shown that differentiation and
maturation mediator causes differentiation of myeloid leukemia H-60 cells
to terminal monocytic cells (2).
Thus, phosphoglucose isomerase is one protein with four unique functions.
It is thought that each of these functions
is controlled by a unique active site (3).
This makes PGI a classic example of a moonlighting protein, a protein that
has more than one independent function (4).
Originally it was estimated that there are 80,000 to 100,000 genes in the
human genome. However, with the completion of the sequence of the
human genome, it is now estimated that there are closer to 30,000 total
genes. One possible explanation for this discrepancy is moonlighting
proteins which perform multiple functions (4).
Other examples of moonlighting proteins found in the literature include
Thrombin, an enzyme that causes blood to clot and functions as a cytokin
(4). Further, Methionine aminopeptidase
removes the amino-terminal methoinine residue from synthesized proteins
and acts as a specific co-factor in translational machinery of the ribosome
(4).
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II. General Structure
Rabbit PG1 is a dimer consisting of two chemically
identical polypeptide chains each composed of 555 amino acids <beta
sheets surrounded by alpha
helicies. Here we show one subunit with
beta
sheets and
alpha
helicies<carboxyl terminus
of each subunit is made up of two helicies and a loop extending into the
adjacent subunit <amino terminus
consists of 36 amino acid residues that form a alpha helix followed by
a loop and a beta sheet <hook, that
could potentially be involved in extracellular function or the attachment
of the subunits, extends from the side of each subunit in a helix-turn-helix
motif <6-phosphgluconate,
an inhibitor <
Reload PGI
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III. Active Site
The active sites,
containing the gluconate 6-phosphate inhibitor,
are located between the two alpha/beta domains at the interface between
subunits one and two <The
crystal structure complex with water molecules indicates that the active
sites are found in the regions lacking water molecules<.
Hydrogen bonds are formed between side chain hydroxyl groups in Ser-159,
Ser-209,
Thr-211,
and Thr-214 and phosphate groups in
gluconate 6-phosphate <.
Further, there is also a hydrogen bond between the phosphate group in gluconate
6-phosphate and a water molecule attached to Thr-217<.
In addition, there are interactions between amine groups of Lys-210
and Thr-211 and a phosphate in the inhibitor
<.
Notably, the previous amino acids are conserved in all known PGI sequences
in mammals, plants, flies, bacteria, and yeast (2).
Reload PGI
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IV. Mechanism of Isomerization
In the proposed mechanism of isomerization, the
cyclic form of glucose 6-phosphate binds to the active site and its ring
oxygen is protonated by the enzyme. This would cause the ring to
open and the C-2 proton of glucose 6-phosphate to become relatively acidic.
His-388
in a
His-388/Glu-216
catalytic diad deprotonates C-2 causing an electron shift that results
in the formation of a double bond between between C-1 and C-2. This
is accompanied by the addition of a proton from Lys-518and
the formation of the cis-enediol intermediate
. Solvent exchange experiments indicate that the same proton that
is removed from C-2 is added back onto C-1 of enediol by His-388.
This causes an electron shift that is accompanied by the removal of a proton
from the oxygen at the C-2 of enediol by Lys-518
and the gain of a proton to the oxygen at C-1 by His-388.
This forms the open structure of fructose 6-phosphate. Next, a
pair of electrons is transferred from the oxygen at C-1 to the C-2, and
the resulting ketose receives a proton from the solvent. This results
in the formation of the ring form of fructose 6phosphate. Arg-272,
also crucial in isomerization, causes glucose 6-phosphate to have a positive
electrostatic potential and stabilizes the negative charges on the enediol
transition state. <
Reload PGI
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V. Moonlighting Capabilities
Recent experiments have confirmed that the neuroleukin
active site is independent of the isomerase active site (2).
It is also thought that autocrine motility factor and a differentiation
and maturation mediator active have unique active sites. Experiments
are in progress to confirm the locations of these independent active sites
(3).
Sequence alignments reveal that there are whole
helices containing non conserved residues. It is likely that non
conserved residues, including the hook, are the result of the the evolution
of a new function (2). As long
as this function does not disrupt isomerization and is beneficial to the
organism, it is likely inherited through natural selection. This
hypothesis is especially possible due to the large size of PGI and its
dimeric structure.
Reload PGI
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VI. Nonspherocytic Hemolytic
Anemia
Mutations in phosphoglucose isomerase are the
third or forth most common cause of nonspherocytic hemolytic anemia, a
disease that cause the cell wall of red blood cells to become fragile (1).
Fragile red blood cell membranes cause both anemia and jaundice.
Nonspherocytic hemolytic anemia is also caused by mutations in triosephosphate
isomerase and pyruvate kinase (2).
Twenty six amino acid mutations in human phosphoglucose isomerase
are known to cause nonspherocytic hemolytic anemia (2).
These mutations are found both inside and outside the active site.
For instance, active site mutations including Ser-277,
Gly-158,
and Arg-272. These
amino acids are
shown in relation to 6-phosphogluconate
bound to the active site <.
Ser-277 is often replaced by lysine which inhibits the formations of hydrogen
bonds and results in protein instability. Arg-272's hydrogen bonding
interactions are crucial in determining the shape of the active site.
Further, a glysine mutation make the active site bulkier and inhibit substrate
binding (2).
Mutations outside the active site include Glu-494
and Thr-223 <
Reload PGI
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VII. References
1. Beutler, Ernest, Carol West, Howard A. Britton,
John Harris, Linda Forman. Glucosephosphate Isomerase (GPI) Deficiency
Mutations Associated with Hereditary Nonspherocytic Hemolytic Anemia (HNSHA).
Blood
Cells, Molecules, and Diseases 23:402-409.
2. Jeffery, Constance J., Brian J. Bahnson, Wade Chaen, and Tagmer
Ring, and Gregory A. Petsko. 2000. Crystal Structure of Rabbit
Phosphoglucose Isomerase, A Glycolytic Enzyme that Moonlights as Neuroleukin,
Autocrin Motility Factor, and Differentiation Mediator. Biochemistry
39:955-964.
3. Personal correspondance with Gregory Petsko: November 2001
4. Petsko, Gregory A. 2001. Size doesn't matter. Genome
Biology 2:1003.1-1003.2.
5. Phosphoglucose Isomerase was downloaded from Protein Database
(1DQR).
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