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Fumarate hydratase (Fumarase)
structure of yeast fumarase.
Available PDB structures:
|1FUO, 1FUP, 1FUQ, 1FUR, 1KQ7, 1VDK, 1YFE, 1YFM, 2FUS, 2ISB, 3E04
Fumarase (or fumarate
hydratase) is an enzyme that catalyzes the reversible
hydration/dehydration of Fumarate
to S-malate. Fumarase comes in two forms: mitochondrial and cytosolic. The
mitochondrial isoenzyme is involved in the Krebs
Cycle (also known as the Citric Acid Cycle), and the cytosolic
isoenzyme is involved in the metabolism of amino acids and
fumarate. Subcellular localization is established by the presence
of a signal sequence on the amino terminus in the mitochondrial
form, while subcellular localization in the cytosolic form is
established by the abence of the signal sequence found in the
This enzyme participates in 3 metabolic pathways: citric acid
cycle, reductive carboxylation cycle (CO2 fixation),
and in renal cell carcinoma.
This enzyme belongs to the family of lyases, specifically the hydro-lyases, which
cleave carbon-oxygen bonds. The systematic name of this enzyme
class is (S)-malate hydro-lyase
(fumarate-forming). Other names in common use include:
- L-malate hydro-lyase
- (S)-malate hydro-lyase
Figure 1: Conversion of fumarate to S-malate.
Figure 2: Conversion of fumarate to S-malate by fumarase through a
Figure 2 depicts the fumarase reaction mechanism. Two acid-base
groups catalyze proton transfer, and the ionization state of these
groups is in part defined by two forms of the enzyme E1
and E2. In E1, the groups exist in an
internally neutralized A-H/B: state, while in E2, they
occur in a zwitterionic A-/BH+ state.
E1 binds fumarate and facilitates its tansformation into
malate, and E2 binds malate and facilitates its
transformation into fumarate. The two forms must undergo
isomerization with each catalytic turnover.
Despite its biological significance, the reaction mechanism of
fumarase is not completely understood. The reaction itself can be
monitored in either direction; however, it is the formation of
fumarate from S-malate in particular that is less understood due to
the high pKa value of the
HR (Fig. 1) atom that is removed without the aid of any
cofactors or coenzymes. However, the
reaction from fumarate to L-malate is better understood, and
involves a stereospecific hydration of fumarate to
produce S-malate by trans-addition of a hydroxyl group and a hydrogen atom through a
trans 1,4 addition of a hydroxyl group. Early research into this
reaction suggested that the formation of fumarate from S-malate
involved dehydration of malate to a carbocationic intermediate,
which then loses the alpha proton to form fumarate. This led to the
conclusion that in the formation of S-Malate from fumarate E1 elimination, protonation of fumarate to
the carbocation was followed by the additional of a hydroxyl group
from H2O. However, more recent trials have provided
evidence that the mechanism actually takes place through an
acid-base catalyzed elimination by means of a carbanionic
intermediate E1CB elimination (Figure
The function of fumarase in the citric acid cycle is to facilitate a
transition step in the production of energy in the form of NADH. In the
cytosol the enzyme functions
to metabolize fumarate, which ends up as a biproduct of the urea cycle as well as
amino acid catabolism. Studies have revealed that the active site
is composed of amino acid residues from three of the four subunits
within the tetrameric enzyme.
The primary binding site on fumarase is known as catalytic site
A. Studies have revealed that catalytic site A is composed of amino
acid residues from three of the four subunits within the tetrameric
enzyme. Two potential acid-base catalytic residues in the reaction
include His 188 and Lys 324.
There are two classes of fumarases.[9
] Classifications depend on the arrangement of
their relative subunit, their metal requirement, and their thermal
stability. These include class I and class II. Class I fumarases
are able to change state or become inactive when subjected to heat
or radiation, are sensitive to superoxide anion, are Iron II (Fe2+)
dependent, and are dimeric proteins consisting of around 120 kD.
Class II fumarases, found in prokaryotes as well as in eukaryotes,
are tetrameric enzymes of 200,000 D that contain three distinct
segments of significantly homologous amino acids. They are also
iron-independent and thermal-stable. Prokaryotes are known to have
three different forms of fumarase: Fumarase A, Fumarase B, and
Fumarase C. Fumarase C is a part of the class II fumarases, whereas
Fumarase A and Fumarase B from Escherichia coli (E.
coli) are classified as class I.
Fumarase deficiency is
characterized by polyhydramnios and fetal brain
abnormalities. In the newborn period, findings include severe
neurologic abnormalities, poor feeding, failure to thrive, and hypotonia. Fumarase
deficiency is suspected in infants with multiple severe neurologic
abnormalities in the absence of an acute metabolic crisis.
Inactivity of both cytosolic and mitochondrial forms of fumarase
are potential causes. Isolated, increased concentration of fumaric acid on urine
organic acid analysis is highly suggestive of fumarase deficiency.
Molecular genetic testing for fumarase deficiency is currently
Fumarase is prevalent in both fetal and adult tissues. A large
percentage of the enzyme is expressed in the skin, parathyroid, lymph, and colon. Mutations in the production and
development of fumarase have led to the discovery of several
fumarase-related diseases in humans. These include benign mesenchymal tumors of the uterus, leiomyomatosis and renal cell carcinoma, and fumarase
deficiency. Germinal mutations in fumarase are associated with
two distinct conditions. If the enzyme has missense mutation and
in-frame deletions from the 3’ end, fumarase deficiency results. If
it contains heterozygous 5’ missense mutation and
deletions (ranging from one base pair to the whole gene), then
leiomyomatosis and renal cell carcinoma/Reed’s syndrome (multiple cutaneous and
uterine leiomyomatosis) could result.[9
Crystal structures of fumarase C from Escherichia
coli have been observed to have two occupied dicarboxylate
binding sites. These are known as the active site and the B
site. The active site and B site are both identified as having
areas unoccupied by a bound ligand. This so-called ‘free’ crystal structure
demonstrates conservation of the active-site water. Similar
orientation has been discovered in other fumarase C crystal
structures. Crystallographic research on the B site of the enzyme
has observed that there is a shift on His129. This information
suggests that water is a permanent component of the active site. It
also suggests that the use of an imidazole-imidazolium conversion
controls access to the allosteric B site.
- ^ a
Based on PDB 1yfm coordinates; Weaver T, Lees M, Zaitsev V, Zaitseva I,
Duke E, Lindley P, McSweeny S, Svensson A, Keruchenko J, Keruchenko
I, Gladilin K, Banaszak L (July 1998). "Crystal structures of
native and recombinant yeast fumarase". J. Mol. Biol.
280 (3): 431–42. doi:10.1006/jmbi.1998.1862. PMID 9665847.
- ^ a
Figure rendered using UCSF Chimera. Molecular graphics images were
produced using the UCSF Chimera package from the Resource for
Biocomputing, Visualization, and Informatics at the University of
California, San Francisco; Pettersen
EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin
TE (October 2004). "UCSF Chimera--a visualization system for
exploratory research and analysis". J Comput Chem
25 (13): 1605–12. doi:10.1002/jcc.20084.
- ^ a
Adrian D. Hegeman; Frey, Perry A.
(2007). Enzymatic reaction mechanisms. Oxford
[Oxfordshire]: Oxford University Press. ISBN
- ^ a
Tadhg P. Begley; McMurry, John (2005).
The organic chemistry of biological pathways. Roberts and
Co. Publishers. ISBN
- ^ a
Walsh C (1979). Enzymatic reaction
mechanisms. San Francisco: W. H. Freeman. ISBN
- ^ a
Estévez M, Skarda J, Spencer J,
Banaszak L, Weaver TM (June 2002). "X-ray crystallographic and
kinetic correlation of a clinically observed human fumarase
mutation". Protein Sci. 11 (6):
1552–7. PMID 12021453. PMC 2373640. http://www.proteinscience.org/cgi/pmidlookup?view=long&pmid=12021453.
c Lynch AM,
Morton CC (2006-07-01). "FH (fumarate
hydratase).". Atlas of Genetics and Cytogenetics in Oncology
and Haematology. http://atlasgeneticsoncology.org/Genes/FHID40573ch1q42.html.
- ^ Weaver T (October 2005). "Structure of
free fumarase C from Escherichia coli". Acta Crystallogr. D
Biol. Crystallogr. 61 (Pt 10): 1395–401. doi:10.1107/S0907444905024194. PMID 16204892.
Mitochondrial enzymes and
citric acid cycle (Citrate
Isocitrate dehydrogenase, Oxoglutarate dehydrogenase,
Succinyl coenzyme A
synthetase, Fumarase, Malate
anaplerotic reactions (Aspartate transaminase, Glutamate dehydrogenase, Pyruvate dehydrogenase
synthetase I, Ornithine transcarbamylase,
Other/to be sorted
I (MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6) - Complex III (MT-CYB) - Complex IV (MT-CO1, MT-CO2, MT-CO3)