Glycolysis
I. Introduction
A. Glycolysis--breakdown of glucose "sweet & splitting"
Also called the Embden-Meyerhof pathway
B. This is an ancient metabolic pathway probably used 3.5 million years ago by the earliest known bacteria. This was prior to the presence of large amount of O2 in the atmosphere. Therefore, the pathway had to function under anaerobic conditions.
1. Glycolysis can take place under aerobic or anaerobic conditions
2. Anaerobic microorganisms derive their metabolic energy from glycolysis
C. Glucose is broken down via a series of 10 reactions to yield two molecules of pyruvate. During this process free energy is released and is conserved in the form of ATP.
D. Principles in its function and regulation are common to all pathways of cell metablism.
E. Universal pathway of glucose catabolism-- present in plants, animals and many microorganisms.
II. General scheme of glycolysis:
A. After reaching pyruvate there are three possible pathways:
1] Anaerobic glycolysis
a) animals--when animal tissues must function anaerobically, such as vigourously contracting skeletal muscle, pyruvate cannot be further oxidized to CO2 and H2O, but is converted to lactate. Some ATP is formed but not as much as by aerobic glycolysis.
glucose ------> 2 lactate-----> diffuses via blood to liver where lactate is converted to pyruvate and ultimately to glucose
b) microorganisms-- anaerobically produce lactic acid: sour milk, sauerkraut, sourdough bread
2] Alcoholic fermentation: pyruvate ----->CO
2 + ethanola) fermentation = anaerobic degradation of glucose or other compounds to various products.
purpose = energy production via ATP
b) living organisms arose from anaerobic atmosphere, therefore this is an ancient type of mechanism of getting ATP from glucose
3] Oxidation via the tricarboxlyic acid cycle to CO2 and H2O
Glycolysis is the first stage of going from glucose to CO2 and H2O in aerobic organisms. Pyruvate from glycolysis ---> TCA cycle for final catabolism.
III. Glycolysis has two phases -- (it is a cytoplasmic pathway)
A. Preparatory phase = Glucose ---> glyceraldehyde -3-P (first 5 steps)
1] 2 molecules of ATP used to prime the the pathway
2] Other 6-carbon sugars such galactose, glucose, mannose, enter the pathway here.
a. Function of 1st phase is to collect the C-chains of all the metabolized hexoses in the form of 1 common product = Glyceraldehyde 3-P
B. Second phase = payoff phase -----> energy is formed when glyceraldehyde 3 P is converted to pyruvate
1] 4 ATP molecules are generated per glucose molecule during this phase of glycolysis
2] 2 ATP's used during phase 1; therefore, a net of 2 ATP's/mole glucose
3] Summary. During glycolysis we have:
a. degradation of carbon skeletons of glucose to pyruvate
b. phosphrylation of ADP to ATP
c. transfer of H+ to NAD+ -----> NADH
IV. Glycolysis takes place via phosphorylated intermediates. There are 9.
There are three functions of the phosphate groups:
A. Ionization at pH 7 and cell impermeablity--
1] Intermediates have a net negative charge
2] Charge prevents molecules from diffusing from the cell
3] Glucose can enter, but cannot diffuse out once phophorylated
B. Energy conversion--essential components of energy conversion.
High energy P is transferrred from inermediate to inermediate.
C. Binding to enzymes--Phosphate groups are binding or recognition sites for binding to enzymes
V. Specific reactions of glycolysis
A. Phosphorylation of glucose-- hexokinase
1] Irreversible under intracellular conditions
2] Mg +2-ATP is the substratefor muscle hexokinase:
3] Other hexoses can be phosphorylated such as D-mannose, D-fructose, but the kM for glucose is lower than that of fructose, so all glucose is used prior to use of fructose.
4] Minor metabolic control reaction, G6P is allosteric inhibitor of hexokinase. When G6P builds up in conc, it feedback inhibits the enzyme to slow down or stop its activity
B. Liver = glucokinase
Glucose + ATP -----> G6P freely reversible reaction
1] Specific for a-D glucose, no activity with other hexoses
2] Not inhibited by G6P
3] Higher KM (lower affinity) for glucose than hexokinase
4] Function: When glucose concentration in the blood is high (after a meal), then glucose is converted to G6P, which is converted to glycogen in the liver (temporary storage form of glucose). If glucose is low, then glycogen is converted to G6P to glucose ---> brain.
5] Diabetics have difficient glucokinase due to lack of insulin . Therefore, blood glucose is high and liver glycogen is low.
B. Glucose- 6- P to Fructose-6-P -- Phosphoglucoisomerase
C. Fructose-6-P to Fructose1,6 diphosphate --phosphofructokinase
1] Reaction is essentially irreversible in cells
2] Major control point of glycolysis
3] Allosterically regulated by ATP and metabolites
a. ATP inhibits activity
b. ADP/AMP stimulates acitivity
c. Citric acid conc increase form TCA-cycle enhances inhibition by ATP
4] Reverse reaction is a different enzyme = fructose-diphosphatase, which is allosterically inhibited by ADP and acitivated by ATP.
D. Fructose 1,6 diphosphate cleavage-- aldolase
1] Reaction has large, positive KM, so that reaction is not favored, but products are rapidly metabolized (hence in low conc), which pulls the reaction toward the left.
2] Enzyme is specific for alcohol groups that are trans
E. Interconversion of triosephosphates --triose phosphate isomerase
Second phase= energy yielding
F. Oxidation of Glyceraldehyde-3-P to 1, 3-bisphosphoglycerate
Glyceraldehyde phosphate dehydrogenase
NADH must be reoxidized for glycolysis to continue--under aerobic conditions NADH ---> ETS 3 ATP's/NADH
G. Transfer of phosphate from 1,3 bisphosphoglycerate to ADP
phosphoglycerate kinase
1] This and proceeding reaction are coupled:
2] This is an example of substrate level phosphorylation.
There are 3 types ofd phosphorylation:
a. Photosynthetic
b. Oxidative ---> ETS
c. Substrate level, where substrate is involved in the process
H. 3-phosphoglycerate to 2-phosphoglycerate
phosphoglycerate mutase
I. Dehydration of 2-phosphoglycerate to phosphoenolpyruvate
enolase
J. Phosphate transfer from PEP to ADP = pyruvate kinase
1] Last step of aerobic glycolysis
2] 3rd control point of glycolysis
3] Inhibited by ATP and citric acid
4] 1/2 the energy of hydrolysis (-14.8 kCal) is recovered as ATP
( - 7.5 kCal) , the rest is the driving force for the reaction.
K. Reduction of pyruvate to lactate
1] In anaerobic tissues NADH cannot be reoxidized unless pyruvate is converted to lactate.
2] LDH has 5 isozyme forms with different kM's for pyruvate and lactate
Alcoholic Fermentation
L. Balance sheet
1] Aerobic
2] Anaerobic
VI. Feeder pathways (see attached sheet for overview)
A. Polysaccharides (starch and glycogen from plants and animals, respectively)
B. Monosaccharides
1] Intestine --fructose
2] Liver --fructose
3] galactose--
C. Disaccharides
VII. Regulation of glycolysis-- Glycogen breakdown
A. Rates of central metabolic pathways that degrade glucose to produce chemical energy as ATP adjust themselves min by min to accomodate needs for ATP. Also regulate intermediates for other aspects of metabolism.
B. Regulation is muscle and liver is different
1. Muscle needs ATP for contraction ---> needs glucose for glycolysis
Regulation of glycogen breakdown is allosterically and hormonally controlled
2. Liver functions to maintain a constant level of glucose in the blood
Regulation of glycogen breakdown is regulated by hormones and blood glucose
C. Glycogen entry ---> regulated by glycogen phosphorylase-a key regulatory enzyme
1] In muscle glycogen phosphorylase is regulated hormonally and allosterically
a] Muscle--> two forms:
phosphorylase a (phosphorylated) -----> active form
phosphorylase b (nonphosphorylated) ----> inactive form
2 ID subunits mw = 190, 000 (94,500 each)
contains essential serine that is phosphorylated
activity in muscle is regulated by the ratio of a/b
b] Phosphorylase a phosphatase removes P from serine
c] Phosphorylase b kinase adds P to serine
2] Glycogen phosphorylase in muscle is regulated in a second way:
a] Phosphorylase b (inactive form )is stimulated by the noncovalent (allosteric) binding of AMP, which increases in the muscle during ATP breakdown. Stimulation by AMP is prevented by ATP, a negative modulator. Actvity of Phosphorylase b reflects the ratio of AMP/ATP
b] Phosphorylase a (active form) is not stimulated by AMP= the AMP-independent form of the enzyme; whereas, phosphorylase b is the AMP-dependent form of the enzyme.
D. Glycogen entry-- hormanal regulation by epinephrine & glucagon --> ultimately hormones regulate the covalent modification of glycogen phosphorylase
1] Hormonal regulation -- occurs both in muscle & liver
a. Epinephrine is secreted by the medulla of the the adrenal gland
b. carried by blood to liver--stimulates the breakdown of glycogen to glucose (also in muscle)
c. Epinephrine binds to receptor sites on the cell surface --> production of cylic AMP, which actvates glycogen phosphorylase, a regulatory enzyme.
E. In liver regulation is hormonal and by glucose
1. Liver glycogen phosphorylase is similar to that in muscle
a] It has the a and b forms
b] Covalent modification is stimulated by glucagon, which is secreted when blood sugar l levels drop.
2. Glycogen in the liver is a reserve of glucose.
a] When blood sugar levels drop below 4 to 5 mM glycogen phosphorylase is activated
b] The G1P is converted to G6P ---> glucose + Pi
G1P -------------> G6P------------------> glucose + Pi
3. Glycogen phosphorylase is also allosterically regulated by glucose
4. Glucokinase
a] Induced only when blood sugar levels are high (not present in significant levels in normal individual
b] Higher kM for glucose than hexokinase (10 mM vs 0.1 mM)
c] No product inhibition
d] Not involved in glycolysis
VIII. Regulation of the Pathway
A. Three major control points: hexokinase, phosphofructokinase and pyruvate kinase
B. Glucose entry ---> First point of entry = hexokinase regulation
1] In some tissues,such as muscle, hexokinase is an allosteric enzyme.
2] If G6P conc in cell builds up indicating that it is not being used as fast as it is formed, then it is inhibited by G6P---> preventing phosphorylation of any more glucose until is is all used up.
3] Glucokinase reaction (liver) is not inhibited by G6P, thus the liver can store large amounts of glycogen, because G6P----> G1P----> glycogen
4] The hormone insulin, secreted by the pancreas into the blood whenever the glucose conc is high, stimulates the synthesis of glucokinase---> glucokinase is deficient in diabetics and during starvation.
C. PFK--complex allosteric enzyme with many stimulatory and inhibitory modulators:
1] Activators = AMP, F-1,6 di P, ADP, phosphate and K+
2] Inhibitors = ATP, citrate Mg2+ and Ca2+
3] ATP and citrate are the most important negative modulators, and AMP and F-1,6,diP are most important positive modulators
4] When ATP is needed PFK activity is accelerated; AMP is potent stimulatory modulator.
5] When ATP is adequate the affinity of PFK for F-6-P drops; this inhibitory effect is enhanced by citrate.
6] PFK is stimulated several hundred-fold when muscle tissue goes form resting to active state.
C. Pyruvate kinase--another control point in glycolysis
1] Another site of metabolic regulation
a] liver enzyme is tetrameric protein , mw 250,000
b] allosterically inhibited by ATP--> high ATP reduces enzyme affinity for ATP
c] activated by 1,6-bis phosphoglycerate
d] Inhibited by acetylCoA derived from long chain FA's
e] synthesis is under dietary control--> high carbo ingestion can increase the levels 10-fold. Carbohydrate loading before athletic event increases levels of this enzyme and may increase the rate of energy generation by glycolysis
2] Regulation by covalent modification--> phosphorylation
D. Notes about the regulation of pathways:
1] Regulated steps of metabolism are usually irreversible under cellular conditions.
2] Most nonregulated steps are at or near equilibrium, but because there are regulated steps, the whole pathway is irreversible.
Pasteur Effect