Phosphoribulokinase (PRK) () is an essential
photosynthetic
Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that, through cellular respiration, can later be released to fuel the organism's activities. Some of this chemical energy is stored in c ...
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
that
catalyzes
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
the
ATP-dependent
phosphorylation
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
of
ribulose 5-phosphate
Ribulose 5-phosphate is one of the end-products of the pentose phosphate pathway. It is also an intermediate in the Calvin cycle.
It is formed by phosphogluconate dehydrogenase, and it can be acted upon by phosphopentose isomerase and phosphopent ...
(RuP) into
ribulose 1,5-bisphosphate
Ribulose 1,5-bisphosphate (RuBP) is an organic substance that is involved in photosynthesis, notably as the principal acceptor in plants. It is a colourless anion, a double phosphate ester of the ketopentose (ketone-containing sugar with five car ...
(RuBP), both
intermediates in the
Calvin Cycle
The Calvin cycle, light-independent reactions, bio synthetic phase, dark reactions, or photosynthetic carbon reduction (PCR) cycle of photosynthesis is a series of chemical reactions that convert carbon dioxide and hydrogen-carrier compounds into ...
. Its main function is to regenerate RuBP, which is the initial
substrate and CO
2-acceptor molecule of the Calvin Cycle.
PRK belongs to the family of
transferase enzymes, specifically those transferring phosphorus-containing groups (
phosphotransferase
Phosphotransferases are a category of enzymes ( EC number 2.7) that catalyze phosphorylation reactions. The general form of the reactions they catalyze is:
:A-P + B \rightleftharpoons B-P + A
Where ''P'' is a phosphate group and A and B are the do ...
s) to an alcohol group acceptor. Along with
ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo), phosphoribulokinase is unique to the Calvin Cycle. Therefore, PRK activity often determines the
metabolic rate
Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run cell ...
in organisms for which
carbon fixation
Biological carbon fixation or сarbon assimilation is the process by which inorganic carbon (particularly in the form of carbon dioxide) is converted to organic compounds by living organisms. The compounds are then used to store energy and as ...
is key to survival.
Much initial work on PRK was done with
spinach
Spinach (''Spinacia oleracea'') is a leafy green flowering plant native to central and western Asia. It is of the order Caryophyllales, family Amaranthaceae, subfamily Chenopodioideae. Its leaves are a common edible vegetable consumed either f ...
leaf extracts in the 1950s; subsequent studies of PRK in other photosynthetic
prokaryotic
A prokaryote () is a Unicellular organism, single-celled organism that lacks a cell nucleus, nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek language, Greek wikt:πρό#Ancient Greek, πρό (, 'before') a ...
and
eukaryotic
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
organisms have followed. The possibility that PRK might exist was first recognized by Weissbach et al. in 1954; for example, the group noted that
carbon dioxide
Carbon dioxide (chemical formula ) is a chemical compound made up of molecules that each have one carbon atom covalently double bonded to two oxygen atoms. It is found in the gas state at room temperature. In the air, carbon dioxide is transpar ...
fixation in crude spinach extracts was enhanced by the addition of ATP.
The first purification of PRK was conducted by Hurwitz and colleagues in 1956.
ATP + Mg
2+ - D-ribulose 5-phosphate
ADP + D-ribulose 1,5-bisphosphate
:
The two
substrates of PRK are
ATP and
D-ribulose 5-phosphate, whereas its two
products
Product may refer to:
Business
* Product (business), an item that serves as a solution to a specific consumer problem.
* Product (project management), a deliverable or set of deliverables that contribute to a business solution
Mathematics
* Produ ...
are
ADP and
D-ribulose 1,5-bisphosphate
Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. It has chemical formula . Two enantiomers are possible, -ribulose (-erythro-pentulose) and -ribulose (-erythro-pentulose). ...
. PRK activity requires the presence of a
divalent
In chemistry, the valence (US spelling) or valency (British spelling) of an element is the measure of its combining capacity with other atoms when it forms chemical compounds or molecules.
Description
The combining capacity, or affinity of an ...
metal
cation
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
like Mg
2+, as indicated in the reaction above.
Structure
The structure of PRK is different in prokaryotes and eukaryotes. Prokaryotic PRK's typically exist as
octamers of 32 kDa
subunits, while eukaryotic PRK's are often
dimers of 40 kDa subunits.
Structural determinations for eukaryotic PRK have yet to be conducted, but prokaryotic PRK structures are still useful for rationalizing the regulation and mechanism of PRK. As of 2018, only two crystal structures have been resolved for this class of enzymes in ''
Rhodobacter sphaeroides
''Rhodobacter sphaeroides'' is a kind of purple bacterium; a group of bacteria that can obtain energy through photosynthesis. Its best growth conditions are anaerobic phototrophy (photoheterotrophic and photoautotrophic) and aerobic chemoheterot ...
'' and ''
Methanospirillum hungatei
In taxonomy, ''Methanospirillum'' is a genus of microbes within the family Methanospirillaceae. All its species are methanogenic archaea. The cells are bar-shaped and form filaments. Most produce energy via the reduction of carbon dioxide with ...
'', with the respective
PDB accession codes an
5B3F
''Rhodobacter sphaeroides''
In ''Rhodobacter sphaeroides'', PRK (or RsPRK) exists as a
homooctomer with
protomer
In structural biology, a protomer is the structural unit of an oligomeric protein. It is the smallest unit composed of at least two different protein chains that form a larger hetero-oligomer by association of two or more copies of this unit.
The ...
s composed of seven-stranded
mixed β-sheets, seven
α-helices
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
, and an auxiliary pair of anti-parallel
β-strands.
The RsPRK subunit exhibits a
protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduci ...
analogous to the folding of
nucleotide monophosphate (NMP) kinases.
Mutagenesis studies suggest that either
Asp
Asp may refer to:
Places
* Asp, part of Densbüren, Aargau, Switzerland
* Aspe (''Asp'' in Valencian), Alicante, Spain
* Asp Lake, a lake in Minnesota
Animals
* Asp (fish)
* Asp (snake), in antiquity, one of several venomous snakes
** ''Cera ...
42 or Asp 169 acts as the
catalytic
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
base that
deprotonates
Deprotonation (or dehydronation) is the removal (transfer) of a proton (or hydron, or hydrogen cation), (H+) from a Brønsted–Lowry acid in an acid–base reaction.Henry Jakubowski, Biochemistry Online Chapter 2A3, https://employees.csbsju.edu ...
the O1
hydroxyl
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
oxygen on RuP for
nucleophilic attack
In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...
of ATP, while the other acts a
ligand
In coordination chemistry, a ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's electr ...
for a metal cation like
Mg2+ (read mechanism below for more details).
Other
residues present at the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
for RsPRK include
His
His or HIS may refer to:
Computing
* Hightech Information System, a Hong Kong graphics card company
* Honeywell Information Systems
* Hybrid intelligent system
* Microsoft Host Integration Server
Education
* Hangzhou International School, in ...
45,
Arg
Arg or ARG may refer to:
Places
*''Arg'' () means "citadel" in Persian, and may refer to:
**Arg, Iran, a village in Fars Province, Iran
**Arg (Kabul), presidential palace in Kabul, Afghanistan
**Arg, South Khorasan, a village in South Khorasan P ...
49, Arg 168, and Arg 173, which are purportedly involved in RuP binding.
(See image at right).
''Methanospirillum hungatei''
In
archaea
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...
l PRK of ''Methanospirillum hungatei,'' PRK (or MhPRK) exists as a
homodimer
In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ''dimer'' ha ...
of two
protomer
In structural biology, a protomer is the structural unit of an oligomeric protein. It is the smallest unit composed of at least two different protein chains that form a larger hetero-oligomer by association of two or more copies of this unit.
The ...
s, each consisting of eight-stranded mixed β-sheets surrounded by α-helices and β-strands—similar to the structure of bacterial PRK from ''R. sphaeroides'' (see info. box above)''.''
Although their
quaternary structures differ and they have low
amino acid sequence identity, MhPRK and RsPRK have structurally similar
N-terminal domains as well as sequentially conserved residues like His 55,
Lys 151, and Arg 154.
Mechanism and Activity
PRK catalyzes the phosphorylation of RuP into RuBP. A catalytic residue in the enzyme (i.e. aspartate in RsPRK) deprotonates the O1 hydroxyl oxygen on RuP and
activates it for nucleophilic attack of the
γ-phosphoryl group of ATP.
As the γ-phosphoryl group is transferred from ATP to RuP, its
stereochemistry
Stereochemistry, a subdiscipline of chemistry, involves the study of the relative spatial arrangement of atoms that form the structure of molecules and their manipulation. The study of stereochemistry focuses on the relationships between stereois ...
inverts.
To allow for such inversion, the catalytic mechanism of PRK must not involve a phosphoryl-enzyme
intermediate.
Some studies suggest that both substrates (ATP and RuP) bind simultaneously to PRK and form a
ternary complex
A ternary complex is a protein complex containing three different molecules that are bound together. In structural biology, ''ternary complex'' can also be used to describe a crystal containing a protein with two small molecules bound, for example ...
. Others suggest that the substrate addition is sequential; the particular order by which substrates are added is still disputed, and may in fact, vary for different organisms.
In addition to binding its substrates, PRK also requires
ligation
Ligation may refer to:
* Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules
* In medicine, the making of a ligature (tie)
* Chemical ligation, the production of peptides from amino acids
* Tubal ligation, a meth ...
to divalent metal cations like
Mg2+ or
Mn2+ for activity;
Hg2+ has been demonstrated to inactivate the enzyme.
Enzyme specificity
PRK shows high
specificity for ribulose 5-phosphate. It does not act on any of the following substrates:
D-xylulose 5-phosphate,
fructose 6-phosphate
Fructose 6-phosphate (sometimes called the Neuberg ester) is a derivative of fructose, which has been phosphorylated at the 6-hydroxy group. It is one of several possible fructosephosphates. The β-D-form of this compound is very common in cells ...
, and
sedoheptulose 7-phosphate
Sedoheptulose 7-phosphate is an intermediate in the pentose phosphate pathway.
It is formed by transketolase and acted upon by transaldolase.
Sedoheptulokinase is an enzyme that uses sedoheptulose and ATP to produce ADP and sedoheptulose 7-phosp ...
.
However, at high
concentration
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', ''number concentration'', an ...
s, PRK may sometimes phosphorylate
ribose 5-phosphate
Ribose 5-phosphate (R5P) is both a product and an intermediate of the pentose phosphate pathway. The last step of the oxidative reactions in the pentose phosphate pathway is the production of ribulose 5-phosphate. Depending on the body's state, r ...
, a compound upstream the
RuBP regeneration step in the Calvin Cycle.
Furthermore, PRK isolated from ''
Alcaligenes eutrophus
''Cupriavidus metallidurans'' is a non-spore-forming, Gram-negative bacterium which is adapted to survive several forms of heavy metal stress.
As a model and industrial system
It is an ideal subject to study heavy metal disturbance of cell ...
'' has been shown to use
uridine triphosphate
Uridine-5′-triphosphate (UTP) is a pyrimidine nucleoside triphosphate, consisting of the organic base uracil linked to the 1′ carbon of the ribose sugar, and esterified with tri-phosphoric acid at the 5′ position. Its main role is as substra ...
(UTP) and
guanosine triphosphate
Guanosine-5'-triphosphate (GTP) is a purine nucleoside triphosphate. It is one of the building blocks needed for the synthesis of RNA during the transcription process. Its structure is similar to that of the guanosine nucleoside, the only diffe ...
(GTP) as alternative substrates to ATP.
pH effects
The phosphorylation reaction proceeds with maximal
velocity
Velocity is the directional speed of an object in motion as an indication of its rate of change in position as observed from a particular frame of reference and as measured by a particular standard of time (e.g. northbound). Velocity is a ...
at
pH 7.9, with no detectable activity at pH's below 5.5 or above 9.0.
Regulation
The
mechanisms by which prokaryotic and eukaryotic PRK's are
regulated
Regulation is the management of complex systems according to a set of rules and trends. In systems theory, these types of rules exist in various fields of biology and society, but the term has slightly different meanings according to context. Fo ...
vary. Prokaryotic PRK's are typically subject to
allosteric regulation
In biochemistry, allosteric regulation (or allosteric control) is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.
The site to which the effector binds is termed the ''allosteric site ...
while eukaryotic PRK's are often regulated by
reversible thiol
In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl gro ...
/
disulfide
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
exchange. These differences are likely due to structural differences in their
C-terminal domains
Allosteric regulation of prokaryotic PRK
NADH
Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an aden ...
is known to stimulate PRK activity, while
AMP #REDIRECT Amp #REDIRECT Amp
{{Redirect category shell, {{R from other capitalisation{{R from ambiguous page ...
{{Redirect category shell, {{R from other capitalisation{{R from ambiguous page ...
(PEP) are known to inhibit activity.
'' PRK.
of PRK.
. Of such metabolites,
has been shown to be the most effective inhibitor of eukaryotic PRK by competing with RuP for the enzyme's active site. This phenomenon may arise from the similarity in
between 6-phosphogluconate and RuP.
More recent work on the regulation of eukaryotic PRK has focused on its ability to form
(G3PDH) or RuBisCo.
'', chloroplast PRK and G3PDH exist as a bi-enzyme complex of 2 molecules of dimeric PRK and 2 molecules of
ic G3PDH thorough association by an Arg 64 residue, which may potentially transfer information between the two enzymes as well.
Multi-enzyme complexes are likely to have more intricate regulatory mechanisms, and studies have already probed such processes. For example, it has been shown that PRK-glyceraldehyde 3-phosphate dehydrogenase complexes in ''
'' only dissociate to release activated forms of its constituent enzymes in the presence of
. Another topic of interest has been to compare the relative levels of PRK activity for when it is complexed to when it is not. For different photosynthetic eukaryotes, the enzyme activity of complexed PRK may be enhanced as opposed to free PRK, and vice versa.
of this enzyme class is ATP:D-ribulose-5-phosphate 1-phosphotransferase. Other names in common use include phosphopentokinase, ribulose-5-phosphate kinase, phosphopentokinase, phosphoribulokinase (phosphorylating), 5-phosphoribulose kinase, ribulose phosphate kinase, PKK, PRuK, and PRK.