Phosphorylase kinase
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Phosphorylase kinase (PhK) is a
serine/threonine-specific protein kinase A serine/threonine protein kinase () is a kinase enzyme, in particular a protein kinase, that phosphorylates the OH group of the amino-acid residues serine or threonine, which have similar side chains. At least 350 of the 500+ human protei ...
which activates
glycogen phosphorylase Glycogen phosphorylase is one of the phosphorylase enzymes (). Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Glycogen phosphory ...
to release
glucose-1-phosphate Glucose 1-phosphate (also called cori ester) is a glucose molecule with a phosphate group on the 1'-carbon. It can exist in either the α- or β-anomeric form. Reactions of α-glucose 1-phosphate Catabolic In glycogenolysis, it is the direct pro ...
from
glycogen Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage in animals, fungi, and bacteria. The polysaccharide structure represents the main storage form of glucose in the body. Glycogen functions as one o ...
. PhK phosphorylates glycogen phosphorylase at two serine residues, triggering a conformational shift which favors the more active glycogen phosphorylase “a” form over the less active glycogen phosphorylase b. The protein is a hexadecameric
holoenzyme 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 product (ch ...
—that is, a homotetramer in which each subunit is itself a tetramer—arranged in an approximate “butterfly” shape. Each of the subunits is composed of an α, β, γ and δ subunit. The γ subunit is the site of the enzyme's catalytic activity while the other three subunits serve regulatory functions. When unmodified, the α and β subunits inhibit the enzyme's catalysis, but
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 both these subunits by
protein kinase A In cell biology, protein kinase A (PKA) is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase (). PKA has several functions in the cell, including regulatio ...
(PKA, or
cAMP Camp may refer to: Outdoor accommodation and recreation * Campsite or campground, a recreational outdoor sleeping and eating site * a temporary settlement for nomads * Camp, a term used in New England, Northern Ontario and New Brunswick to descri ...
-dependent protein kinase) reduces their respective inhibitory activities. The δ subunit is the ubiquitous eukaryotic protein calmodulin which itself has 4 calcium ion binding sites. When cytosolic Ca2+ levels rise-to as low as 10−7 M—the δ subunit undergoes a large conformational change that activates the kinase's activity by binding to a complementary
hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, th ...
patch on the catalytic γ subunit.


Genes

* Alpha: PHKA1, PHKA2 * Beta: PHKB * Gamma: PHKG1, PHKG2


History

Phosphorylase kinase was the first protein kinase to be isolated and characterized in detail, accomplished first by Krebs, Graves and Fischer in the 1950s. At the time, the scientific community was largely unaware of the importance of protein phosphorylation in the regulation of cellular processes, and many in the field dismissed phosphoproteins as biologically unimportant. Since covalent modification by phosphorylation is a widespread, important method of biochemical regulation in a wide variety of cellular processes, the discovery of this reaction has had enormous impact on scientific understanding of regulatory mechanisms. The substrate of PhK, glycogen phosphorylase, had been isolated by Carl and Gerty Cori in the 1930s, who determined that there were two forms: an inactive form b and an active form a. However, for unknown reasons at the time, the only way to isolate glycogen phosphorylase a from muscle tissue was by paper filtration – other methods, such as centrifugation, would not work. It was a critical insight on the part of Fischer et al. that it was the presence of calcium ions in the filter paper that was generating the active “a” isoform. Later research revealed that the calcium ions were in fact activating phosphorylase kinase via the δ regulatory subunit, leading to the phosphorylation of glycogen phosphorylase.


Mechanism

The precise details of the PhK’s catalytic mechanism are still under study. While this may seem surprising given that it was isolated over 50 years ago, there are significant difficulties in studying the finer details of PhK’s structure and mechanism due to its large size and high degree of complexity. In the active site, there is significant homology between PhK and other so-called P-loop protein kinases such as protein kinase A (PKA, cAMP-dependent kinase). In contrast to these other proteins, which typically require phosphorylation of a serine or tyrosine residue in the
catalytic site In biology and biochemistry, the active site is the region of an enzyme where Enzyme substrate, substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid, amino acid residues that form temporary bonds with t ...
to be active, the catalytic γ subunit of PhK is constitutively active due to the presence of a negatively charged glutamate residue, Glu-182. Structural and biochemical data suggest one possible mechanism of action for the phosphorylation of glycogen phosphorylase by PhK involves the direct transfer of
phosphate In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid . The phosphate or orthophosphate ion is derived from phospho ...
from
adenosine triphosphate Adenosine triphosphate (ATP) is an organic compound that provides energy to drive many processes in living cells, such as muscle contraction, nerve impulse propagation, condensate dissolution, and chemical synthesis. Found in all known forms of ...
(ATP) to the substrate
serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...
.


Structure

Phosphorylase kinase is a 1.3 MDa hexadecameric holoenzyme, though its size can vary somewhat due to substitution of different subunit isoforms via mRNA splicing. It consists of four homotetramers each comprised four subunits (α,β,δ,γ). Only the γ subunit is known to possess catalytic activity, while the others serve regulatory functions. Due to the instability of the regulatory subunits in solution, only the γ subunit has been crystallized individually: Overall, the subunits are arranged in two lobes oriented back-to-back in what has been described as a “butterfly” shape with D2 symmetry. Each lobe consists of two tetramers, each consisting of the αβδγ subunits as described earlier. The δ subunit is indistinguishable from cellular
calmodulin Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the bind ...
, while the α and β subunits are close homologues of each other which are proposed to have arisen by
gene duplication Gene duplication (or chromosomal duplication or gene amplification) is a major mechanism through which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gene. ...
and subsequent differentiation.


Biological function and regulation

Physiologically, phosphorylase kinase plays the important role of stimulating glycogen breakdown into free glucose-1-phosphate by phosphorylating glycogen phosphorylase and stabilizing its active conformation. This activity is particularly important in liver and muscle cells, though for somewhat different purposes. While muscle cells generally break down glycogen to power their immediate activity, liver cells are responsible for maintaining glucose concentration in the bloodstream. Thus, the regulatory mechanisms of PhK activity vary somewhat depending on cell type. In general, the enzyme is regulated
allosterically 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 sit ...
and by reversible phosphorylation. Hormones, nerve impulses and muscle contraction stimulate the release of calcium ions. These act as an
allosteric 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 ...
activator, binding to the δ subunits of phosphorylase kinase, and partly activating enzyme activity. This binding partly stabilizes the protein in the active form. The phosphorylase kinase is completely activated when the β and α subunits are phosphorylated by protein kinase A and the delta subunit has bound to calcium ions. In muscle cells, phosphorylation of the α and β subunits by PKA is the result of a cAMP-mediated cell
signaling cascade A biochemical cascade, also known as a signaling cascade or signaling pathway, is a series of chemical reactions that occur within a biological cell when initiated by a stimulus. This stimulus, known as a first messenger, acts on a receptor that ...
initiated by the binding of
epinephrine Adrenaline, also known as epinephrine, is a hormone and medication which is involved in regulating visceral functions (e.g., respiration). It appears as a white microcrystalline granule. Adrenaline is normally produced by the adrenal glands and ...
to
β-adrenergic The adrenergic receptors or adrenoceptors are a class of G protein-coupled receptors that are targets of many catecholamines like norepinephrine (noradrenaline) and epinephrine (adrenaline) produced by the body, but also many medications like beta ...
receptors on the cell surface. Additionally, the release of calcium ions from the
sarcoplasmic reticulum The sarcoplasmic reticulum (SR) is a membrane-bound structure found within muscle cells that is similar to the smooth endoplasmic reticulum in other Cell (biology), cells. The main function of the SR is to store calcium ions (Ca2+). Calcium in bio ...
during muscle contraction inactivates the inhibitory δ subunit and activates PhK fully. In liver cells, the process is somewhat more complex. Both glucagon and epinephrine can trigger the cAMP-PKA cascade, while epinephrine also binds to the
α-adrenergic The adrenergic receptors or adrenoceptors are a class of G protein-coupled receptors that are targets of many catecholamines like norepinephrine (noradrenaline) and epinephrine (adrenaline) produced by the body, but also many medications like beta ...
receptor to trigger a phosphoinositide cascade, resulting in the release of Ca2+ from the
endoplasmic reticulum The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum ( ...
. When the cell needs to stop glycogen breakdown, PhK is dephosphorylated by protein
phosphatases In biochemistry, a phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol. Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases. ...
1 and 2, returning the α and β subunits to their initial inhibitory configuration.


Relation to disease

Defects in phosphorylase kinase genes are the cause of
glycogen storage disease type IX Glycogen storage disease type IX is a hereditary deficiency of glycogen phosphorylase kinase B that affects the liver and skeletal muscle tissue. It is inherited in an X-linked or autosomal recessive manner. update 2011 Signs and symptoms Th ...
(GSD type IX) and GSD type VI (formerly GSD type VIII), which can affect the liver and/or muscles. Among these gene defects, some of the most common are the X-linked liver glycogenosis (XLG) diseases, which can be subdivided into XLG I and XLG II. Clinically, these diseases manifest in slow childhood body development and abnormal enlargement of the liver. In XLG I, PhK activity is abnormally reduced in both blood cells and liver cells, while in XLG II enzyme activity is diminished only in liver cells. These diseases are both due to mutations in the PHKA2 gene, which codes for the α subunit of phosphorylase kinase. In the case of XLG I, mutations are often
nonsense mutations In genetics, a nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a ''nonsense codon'' in the transcribed mRNA, and in leading to a truncated, incomplete, and usually nonfunctional protein produc ...
which result in malformed, unstable α subunits, while mutations in XLG II tend to be
missense In genetics, a missense mutation is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution. Substitution of protein from DNA mutations Missense mu ...
changes which alter the subunits less severely. Based on bioinformatic and structural data, some have suggested that the α and β subunits may have catalytic activity similar to glycoamylases, and that missense mutations in these regions of the α subunit may contribute to the symptoms of XLG II. However, this proposed catalytic activity has yet to be proven directly.


See also

*
calmodulin Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the bind ...
*
glycogenolysis Glycogenolysis is the breakdown of glycogen (n) to glucose-1-phosphate and glycogen (n-1). Glycogen branches are catabolized by the sequential removal of glucose monomers via phosphorolysis, by the enzyme glycogen phosphorylase. Mechanism The ...


References


External links

* *
Overview at ox.ac.uk
{{Portal bar, Biology, border=no Protein kinases EC 2.7.11