Biochemistry
Seventh Edition
Glycogen Metabolism
OUTLINE
Glycogen breakdown requires the interplay of several enzymes Phosphorylase is regulated by allosteric interactions and reversible phosphorylation Epinephrine and glucagon signal the need for glycogen breakdown Glycogen is synthesized and degraded by different pathways Glycogen breakdown and synthesis are reciprocally regulated
Glycogen
Glycogen is a highly branched, very large polymer of glyc mols linked 1 4 Branches arise by 1 6 at about every 8-10th residue It is found in the cytosol. It is the storage form of Glc. Liver and muscle are the major sites for the storage of glycogen.
Degradation of Glycogen
Is not a simple reversal of the synthetic pathway Other enzymes involved Glycogen G-1-P Shortening of chains: Glycogen phosphorylase (-1 4) It is an exoglucosidase Degrades the gly. chains at their non-reducing ends until four glucosyl units remain on each chain before the branch point The resulting structure a limit dextrin Phosphorylase cannot degrade this any further!
Branches are removed through two enzymatic activities of the debranching enzyme
a. b. Glucosyl 4:4 transferase removes the outer 3 of 4 glucosyl residues Single glucose residue attached in an 16 linkage is then removed by the -amylo (1:6) glucosidase activity of the debranching enzyme, releasing free glucose
Regulation phosphorylase
Regulation of glycogen metabolism is different in muscle and liver.
In muscle, the end served by glycolysis is ATP production and the rate of glycolysis increases as muscle works more, demanding more ATP. The liver has a different role in whole-body metabolism and glucose metabolism in the liver is different. The liver makes sure that glucose level is constant in the blood, producing and exporting Glc.
Muscle phosphorylase
2 subunits, in each Ser residue at position 14 is Plated (Phosphorylase kinase does this)
2.
Phosphorylase b:
inactive form in resting muscle, all enzyme is its inactive form
structurally identical except that Ser residues are not Plated. It is active when AMP is high! It is inactive when ATP and Glc 6-P are high! So, muscle phosphorylase b is active only when the energy charge of the muscle is low.
The rate of glycogen breakdown is due to the a/b which is controlled by hormones especially by epinephrine.
Phosphorylase a phosphorylase b by dephosphorylation catalyzed by phosphorylase a phosphatase.
Muscle phosphorylase
Both phosphorylase b and phosphorylase a exist as equilibria between an active R state and a less active T state. Phosphorylase b is usually inactive because the equilibrium favors the T state.
Phosphorylase a is usually active because the equilibrium favors the R state.
Intensive exercise AMP/ATP goes up, Phosphorylase b (active) In resting muscle AMP/ATP goes down, Phosphorylase b (inactive)
Exercise will also result in hormone release (epinephrine) that generates the phosphorylated a form of the enzyme.
Liver phosphorylase
Liver phosphorylase and muscle phosphorylase are 90% identical in amino acid sequence. Liver phosphorylase a, but not b, has the most responsive T-to-R transition. The binding of Glc shifts the allosteric equilibrium of the a form from the R to the T state, deactivating the enzyme. Why would Glc function as a negative regulator of liver phosphorylase a?
When there is plenty of Glc, no need to breakdown liver glycogen!
Has a fully active form and an inactive form Has a mass of 1200 kd Consist of 4 subunits (bgd)
The subunit g is the source of catalytic activity. The other subunits are regulatory subunits.
Phosphorylase kinase has the highest activity only after both the phosphorylation of the b subunit and the activation of the d subunit by Ca binding.
PKA activates phosphorylase kinase Glycogen phosphorylase activated Glc 1-P is made What activates PKA?
HORMONES
Glucagon Epinephrine
Signal transduction
Epi GTP-bound G proteins Increased cAMP PKA increases
UDPG synthesis:
G-6-P G-1-P G-1-P + UTP UDPG + PPi
B.
2.
A fragment of glycogen can serve as a primer in cells whose glycogen stores are not totally depleted. If a glycogen fragment is not present, glycogenin, a glycosyltransferase, serves as the primer.
If no other enzyme acts on the chain, the resulting structure is a linear molecule of glucosyl residues attached by 1-4.
Such a compound, called amylose, is found in fruits.
The UDP released when the new bond is made can be convert back. UDP + ATP UTP + ADP
This enzyme transfers 5 to 8 glucosyl residues from the nonreducing end to another residue by an 1,6 link. Further elongation Branches have two important functions
a) increases the solubility of the glycogen molecule. b) The number of non-reducing ends to which new glucosyl residues can be added and thereby greatly accelerating the rate at which glycongen synthesis and degradation can occur.
UDP-Glucose synthesis UDP-glucose phosphorylase A primer is required for glycogen synthesis (glycogenin or a fragment of glycogen) Glc units are added to the either the existing glycogen chains or glycogenin (enzyme glycogen synthase).
C-4 is the non-reducing end of glycogen chain. New glucose molecules are always added to this non-reducing terminus.
Elongation of glucose chains Creating branches in glycogen (enzyme transferase) Branches have 2 functions:
1. Increase the solubility of the glycogen molecule 2. Increase the rate of glycogen synthesis
Glucagon and Epi promote glycogenolysis, at the same time they inhibit glycogen synthesis.
Both effects are mediated by cAMP and cAMP dependent protein kinase. Regulated enzyme: glycogen synthase
a form: active (not phosphorylated) b form: inactive (phosphorylated) PKA and other kinases phosphorylate the enzyme. Protein kinase (Ser phosphorylated)
Inactive forms are shown in red, active forms are shown in green.
Glycogen degradation
Inactive forms are shown in red, active forms are shown in green.
Glycogen synthesis
Inactive forms are shown in red, active forms are shown in green.
Hormone -triggered cAMP cascade acting through PKA Glycogen breakdown and synthesis are reciprocally regulated.
Phosphorylase kinase also inactivates glycogen synthase.
Thus, Epi increases glycogen breakdown by making phosphorylase a and decreases glycogen synthesis by making inactive phosphatase.
When blood glucose is high, insulin is stimulated. Activated insulin-sensitive protein kinase makes activated protein phosphatase
The consequent dephosphorylation of glycogen synthase, phosphorylase kinase, and phosphorylase promotes glycogen synthesis and blocks its degradation!
Phosphorylated groups can be removed by phosphatases; therefore, the action of phosphatases always opposes kinases.
If kinases activity is greater than activity of phosphatase, the enzyme is in the phosphorylated mode.
Insulin, Glucagon, and Epi are three important hormones which affect glycogen metabolism!
After a carbohydrate-rich meal blood glucose increases. Insulin is the primary signal for glycogen synthesis. Blood glucose level 80-120 mg/dL (4.4-6.7 mM) The liver senses the concentration of blood glucose and either release or takes up glucose.
Phosphorylase a is the glucose sensor in liver cells Glucose is high Binding of Glc converts R T PP1 is released Inactivation of glycogen breakdown and the activation of glycogen synthesis take place..
Key enzymes are phophorylated by a family of kinases, some of which are cAMP dependent. Phosphorylation of an enzyme causes 3D change that affects the active site. It may either increase or decrease its activity depending on the type of enzyme.
There are 8 glycogen storage diseases but we will only cover Type I and Type V
Type I
Von Gierke Disease Glc 6-phosphatase is missing.
Type V
McArdle Disease Phosphorylase is missing.
Problem 31. Purified from two samples of human liver, glycogen was either treated or not treated With a-amylase and subsequently analyzed by SDS-PAGE and western blotting with Berg Tymoczko Stryer the use of antibodies to glycogenin. The results are presented in next slide. 1. Why are no proteins without amylase?
Glycogen is too large to enter the gel. Antibody to glycogenin was used so we only see Glycogenin by western blotting.
Biochemistry
Glygogen Metabolism
2. 3.
Seventh Edition What is the reason using amylase? Amylase degrades glycogen, releases glycogenin Why dont we see other proteins? 1. Antibody against glycogenin was used21 (glycogen P-lase, glycogen synthase, CHAPTER Protein phosphatase-1 can be seen if we used Abs for them)
Problem for students. The gene for glycogenin was transfected into a cell line that normally stor small amounts of glycogen. The cells were then manipulated according to the following protocol, and glycogen was isolated and analyzed by SDS-PAGE and western blotting by using An antibody to glycogenin with and without amylase treatment. The results are presented in the Berg Tymoczko Stryer next slide. The protocol: Cells cultured in growth medium and 25 mM glucose (lane1) were Switched to medium containing no glucose for 24 hours (lane 2). Glucose-starved cells were refed with medium containing 25 mM glucose for 1 hour (lane 3) or 3 hours (lane 4). Samples (12 microg of protein) were either treated or not treated with amylase, before being loaded on the gel. a. Why did the western analysis produce a smear-that is, the high molecular-weight staining in lane 1(-)? Seventh Edition b. What is the significance of the decrease in HMW-staining in lane 2(-)? c. What is the significance of the difference between lanes 2(-) and 3(-)? d. Suggest a plausible reason why there is essentially no difference between lanes 3(-) and 4(-)? CHAPTER 21 e. Why are the bands at 66 kd the same in the lanes treated with amylase, despite the Glygogen Metabolism fact that the cells were treated differently?
Biochemistry