Phosphorylase kinase
Encyclopedia
Phosphorylase kinase is a serine/threonine-specific protein kinase
which activates glycogen phosphorylase
to release glucose-1-phosphate
from glycogen
. 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 homotetramer of tetramers arranged in an approximate “butterfly” shape. Each of the four tetramers 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
of both these subunits by protein kinase A (PKA, or cAMP
-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 patch on the catalytic γ subunit.
The substrate of PhK, glycogen phosphorylase, had been isolated by Carl and Gerty Cori in the 1930’s, 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.
Structural and biochemical data suggest one possible mechanism of action for the phosphorylation of glycogen phosphorylase by PhK involves the direct transfer of phosphate
from adenosine triphosphate
(ATP) to the substrate serine
.
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
, while the α and β subunits are close homologues of each other which are proposed to have arisen by gene duplication
and subsequent differentiation.
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 and by reversible phosphorylation. Hormones, nerve impulses and muscle contraction stimulate the release of calcium ions. These act as an allosteric 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 initiated by the binding of epinephrine
to β-adrenergic receptors on the cell surface. Additionally, the release of calcium ions from the sarcoplasmic reticulum 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 receptor to trigger a phosphoinositide cascade, resulting in the release of Ca2+ from the endoplasmic reticulum
.
When the cell needs to stop glycogen breakdown, PhK is dephosphorylated by protein phosphatases 1 and 2, returning the α and β subunits to their initial inhibitory configuration.
Serine/threonine-specific protein kinase
Serine/threonine protein kinases phosphorylate the OH group of serine or threonine .At least 125 of the 500+ human protein kinases are serine/threonine kinases .-Regulation:...
which activates glycogen phosphorylase
Glycogen phosphorylase
Glycogen phosphorylase is one of the phosphorylase enzymes . Glycogen phosphorylase catalyzes the rate-limiting step in the degradation of glycogen in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond...
to release glucose-1-phosphate
Glucose-1-phosphate
Glucose 1-phosphate is a glucose molecule with a phosphate group on the 1'-carbon.-Catabolic:In glycogenolysis, it is the direct product of the reaction in which glycogen phosphorylase cleaves off a molecule of glucose from a greater glycogen structure.To be utilized in cellular catabolism it must...
from glycogen
Glycogen
Glycogen is a molecule that serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue...
. 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 homotetramer of tetramers arranged in an approximate “butterfly” shape. Each of the four tetramers 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
Phosphorylation
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
of both these subunits by protein kinase A (PKA, or cAMP
Cyclic adenosine monophosphate
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
-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 patch on the catalytic γ subunit.
History
Phosphorylase kinase was the first protein kinase to be isolated and characterized in detail, accomplished first by Krebs, Graves and Fischer in the 1950’s. 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 1930’s, 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 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
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
from adenosine triphosphate
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
(ATP) to the substrate serine
Serine
Serine is an amino acid with the formula HO2CCHCH2OH. It is one of the proteinogenic amino acids. By virtue of the hydroxyl group, serine is classified as a polar amino acid.-Occurrence and biosynthesis:...
.
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
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
, while the α and β subunits are close homologues of each other which are proposed to have arisen by gene duplication
Gene duplication
Gene duplication is any duplication of a region of DNA that contains a gene; it may occur as an error in homologous recombination, a retrotransposition event, or duplication of an entire chromosome.The second copy of the gene is often free from selective pressure — that is, mutations of it have no...
and subsequent differentiation.
Biological function and regulation
Physiologically, phosphorylase kinase plays the important role of stimulating glycogen breakdown into free glucoseGlucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
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 and by reversible phosphorylation. Hormones, nerve impulses and muscle contraction stimulate the release of calcium ions. These act as an allosteric 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 initiated by the binding of epinephrine
Epinephrine
Epinephrine is a hormone and a neurotransmitter. It increases heart rate, constricts blood vessels, dilates air passages and participates in the fight-or-flight response of the sympathetic nervous system. In chemical terms, adrenaline is one of a group of monoamines called the catecholamines...
to β-adrenergic receptors on the cell surface. Additionally, the release of calcium ions from the sarcoplasmic reticulum 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 receptor to trigger a phosphoinositide cascade, resulting in the release of Ca2+ from the endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
.
When the cell needs to stop glycogen breakdown, PhK is dephosphorylated by protein phosphatases 1 and 2, returning the α and β subunits to their initial inhibitory configuration.