REGULATION OF GLYCOLYSIS

THE CONTINUATION

G of Glycolysis Reactions
Seven out of ten catalytic reactions are nearequilibrium because the G of reaction under cellular condition is near zero. Small changes in the concentrations of reactants and products could push any of these reactions either forward or backward. The enzymes that catalyze these reactions are called nonregulatory enzymes.
Actual Free Energy Change of Glycolysis Reactions
Biochemistry, 4 Edition edited by Reginald H. Garrett and Charles M. Grisham 2010 Books/Cole

G of Glycolysis Reactions
Reactions 1, 3, and 10 are nonequilibrium because they exhibit large - G values under cellular conditions. Hence, they are the sites of glycolytic regulation. The enzymes that catalyze these reactions are called regulatory enzymes. If the regulatory enzymes are active, glycolysis proceeds. If the regulatory enzymes are inactive, glycolysis stops.
Actual Free Energy Change of Glycolysis Reactions
Biochemistry, 4 Edition edited by Reginald H. Garrett and Charles M. Grisham 2010 Books/Cole

Mechanisms on Altering the Flux through the Regulated Sites

1. Allosteric Control Substrates, products, or coenzymes in the pathway act as allosteric effectors on the activity of the enzyme that regulates the flux; not the enzyme directly regulating the flux.

2. Covalent Modification (Enzymatic Conversion) The activity of the enzyme that regulates the flux is altered by enzymatic phosphorylation or dephosphorylation at specific sites (e.g. Ser, Thr, or Tyr residues).

Mechanisms on Altering the Flux through the Regulated Sites

3. Substrate Cycles Substrate cycles are two opposing nonequilibrium reactions (reversible-like reactions) that are catalyzed by different enzymes. They are more sensitive to the concentrations of allosteric effectors than is the flux through a single unopposed nonequilibrium reaction.

4. Genetic Control Enzyme concentrations and hence enzyme activities may be altered by protein synthesis in response to metabolic needs.

Phosphofructo#2 6-Phosphofructo-1-kinase (PFK-1) (PFKKey Regulatory Enzyme of Glycolysis

6-phosphofructo-1kinase (PFK-1) catalyzes the first committed step of glycolysis because a. the reaction catalyzed by phosphoglucose isomerase is reversible b. cells make use of G6P in the pentose phosphate pathway and glycogen synthesis

Priming Stage of the Glycolytic Pathway

Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Figure 15.11 Important regulatory features of glycolysis.

Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Allosteric Regulation of PFK-1 PFKNegative Allosteric Effectors ATP Citrate Hydrogen ions Positive Allosteric Effectors AMP/ADP Fructose 2,6-biphosphate energy state of the cell insulin/glucagon ratio in the blood Changes Signalled by Allosteric Effectors energy state of the cell availability of alternate fuels such as fatty acids and ketone bodies internal environment of the cell

Structure of PFK-1 PFK6-Phosphofructo-1-kinase (PFK-1) is a 340-kd tetramer allosteric enzyme that belongs to the family of phosphotransferases. It catalyzes the transfer of phosphate from ATP to fructose 6-phosphate (F6P).

Structure of Bacillus Stearothermophilus 6-Phosphofructo-1-kinase

www.rcsb.org/pbd

Structure of PFK-1 PFKPFK-1 has two conformational states, a high-affinity R state (actived state) and a low-affinity T state (inhibited state), that are in equilibrium.

a
(a) Activated R state of PFK-1, 4PFK.pbd
www.rcsb.org/pbd

b
(b) Inhibited T state of PFK-1, 6PFK.pdb

Structure of PFK-1 PFKEach subunit has two binding sites: the catalytic site (enclosed in light blue) and the regulatory site (enclosed in light green).

Binding Sites in 6-Phosphofructo-1-kinase Subunits

Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Structure of PFK-1 PFK-

The substrate fructose 6-phosphate (F6P) preferentially binds at the catalytic site in the R state.

Binding Sites in 6-Phosphofructo-1-kinase Subunits

Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Structure of PFK-1 PFKAllosteric activators of PFK1 bind at the regulatory site in the R state.

Allosteric inhibitors of PFK1 bind at the regulatory site in the T state.

Binding Sites in 6-Phosphofructo-1-kinase Subunits

Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Structure of PFK-1 PFKSince ATP is both a substrate and an inhibitor of PFK-1, each subunit of PFK-1 has two distinct binding sites: a substrate site (at the catalytic site) and an inhibitor site (at the regulatory site). The substrate site binds ATP equally well in either R or T state but the inhibitor site binds ATP almost exclusively in the T state.
Binding Sites in 6-Phosphofructo-1-kinase Subunits
Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Side View of PFK-1 Structure PFK-

E. Coli 6-Phosphofructo-1-kinase
Lehninger Principles of Biochemistry, 5th Edition edited by David L. Nelson and Michael M. Cox 2008 W.H. Freeman and Company

Top View of PFK-1 Structure PFK-

Structure of E. Coli 6-Phosphofructo-1-kinase

Biochemistry, 7th Edition edited by eremy M. Berg, John L. Tymoczko and Lubert Stryer 2012 W.H. Freeman and Company

Structural Basis for the Allosteric Regulation of PFK-1 PFK-

Fragments of the T-state (blue) and R-state (red) undergo a large conformational rearrangement on the T to R allosteric transition (indicated by the arrows). Residues of the R-state structure are marked by a prime.
Conformational Change in 6-Phosphofructo-1-kinase Enzyme
Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Structural Basis for the Allosteric Regulation of PFK-1 PFK-

In the R state, the side chain (+) of Arg 162 forms a salt bridge with the phosphoryl group (-) of F6P bound in the active site of another subunit.
Conformational Change in 6-Phosphofructo-1-kinase Enzyme
Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Structural Basis for the Allosteric Regulation of PFK-1 PFK-

On the transition of R to T, Arg 162 located at the end of the helical turn unwinds. Thereby, the positively charged side chain of Arg 162 swings away and is replaced by the negatively charged side chain of Glu 161.
Conformational Change in 6-Phosphofructo-1-kinase Enzyme
Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Regulation of PFK-1 by ATP PFK-

At Low [ATP] ATP acts as a substrate ATP binds only at the catalytic site R state of PFK1 PFK1s affinity for F6P is increased Glycolysis turns on

At High [ATP] ATP acts as a heterotropic allosteric inhibitor ATP binds not only in the catalytic site but also in the regulatory site T state of PFK1 PFK1s affinity for F6P is decreased Glycolysis turns off

Regulation of PFK-1 by ATP PFK-

The hyperbolic curve (black) of PFK-1s activity vs [F6P] at low [ATP] shows the noncooperative binding of ATP to PFK-1. The sigmoidal curve (red) of PFK-1s activity vs [F6P] at high [ATP] shows the negative cooperative binding of ATP to PFK-1.
PFK1 Activity vs [F6P] Curves at low [ATP] and high [ATP]
Lehninger Principles of Biochemistry, 5th Edition edited by David L. Nelson and Michael M. Cox 2008 W.H. Freeman and Company

Regulation of PFK-1 by ATP PFK-

At a constant [F6P] (marked with dash line), PFK-1s activity at low [ATP] is higher than PFK-1s activity at high [ATP]. This implies that glycolysis is faster at low [ATP] and glycolysis is slower at high [ATP].

PFK1 Activity vs [F6P] Curves at low [ATP] and high [ATP]

Lehninger Principles of Biochemistry, 5th Edition edited by David L. Nelson and Michael M. Cox 2008 W.H. Freeman and Company

Pasteur Effect

Pasteur Effect refers to the inhibition of glucose utilization and lactate accumulation that occurs when respiration is initiated in anaerobic cells. In the Absence of O2 (Anaerobic Condition) Pyruvate is converted to lactate or ethanol by fermentation. D-glucose + 2ADP3- + 2Pi22L-lactate- + 2ATP4In the Presence of O2 (Aerobic Condition) Pyruvate is converted to carbon dioxide by cellular respiration. (Krebs cycle or TCA cycle) D-glucose + 6O2 + 32ADP3- + 32Pi2- + 32H+ 6CO2 + 6H2O + 32ATP4-

Pasteur Effect

Pasteur Effect refers to the inhibition of glucose utilization and lactate accumulation that occurs when respiration is initiated in anaerobic cells. In the Absence of O2 (Anaerobic Condition) Pyruvate is converted to lactate or ethanol by fermentation. D-glucose + 2ADP3- + 2Pi22L-lactate- + 2ATP4In the Presence of O2 (Aerobic Condition) Pyruvate is converted to carbon dioxide by cellular respiration. (Krebs cycle or TCA cycle) D-glucose + 6O2 + 32ADP3- + 32Pi2- + 32H+ 6CO2 + 6H2O + 32ATP4-

This means that facultive anaerobic cells are provided with oxygen, causing the glycolysis rate to slow down and allowing complete oxidation of glucose into CO2 and H2O, bypassing lactate formation.

Experimental Observation: A small change in ATP concentration accounts for large changes in flux through PFK-1. Example: The ATP concentration in muscle during a vigorous exercise is only about 10% lower than that during the resting state. However, the difference in the rates of glycolysis (during the resting state and during a vigorous exercise) is large. The Argument: A small decrease in ATP concentration is not directly accountable for the large change in metabolic flux.

If its not ATP then what?

Adenylate kinase (AK)



B


catalyzes the transfer of one phosphodiester bond to another in this reaction

2 ADP

ATP + AMP

maintains the equilibrium concentrations of adenine nucleotides in the cytosol

Keq = [ATP] [AMP] [ADP]

intracellular concentration of AMP:

[AMP] = Keq [ADP] [ATP]

A small decrease in [ATP] leads to a greater percentage increase in [AMP] than in the percentage decrease in [ATP].

...then it is

AMP.

Regulation of PFK-1 by AMP PFK-

A PFK-1 solution containing 1 mM ATP and 0.5 mM F6P is brought to 0.1 mM in AMP. The activity of PFK rises from 10% to 50% of its maximal activity that is a 5-fold increase. (in red curve)

PFK-1 Activity versus F6P Concentration

Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Comparison Between AK and other kinases

Other Kinases Adenylate kinase specific phosphoryl-transfer specific phosphoryl-transfer reaction reaction driven by exergonic free energy driven by neutral free energy (equilibrium reaction)

Mechanism: Mechanism: closing of jaws on the energetic bound substrates and counterweight opening of jaws when the product is formed whole structure undergoes a conformational change only the remote domains undergo conformational change

Mechanism of (AK)

On substrate binding, a portion of the adenylate kinase remote from the active site (cyan and blue domains) increases its chain mobility and thereby absorbs some of the free energy of substrate binding.

This mechanism acts as an energetic counterweight that permits facile product release and hence maintains a high reaction rate.
Conformational Change in E. Coli Adenylate kinase
Biochemistry, 4th

a. liganded

b. unliganded

Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Law of Supply and Demand

high glucose requirement low ATP generation low glucose requirement high ATP generation

S U M M A R Y

Low Energy Status PFK1 activity is enhanced

High Energy Status PFK1 activity is inhibited

AMP/ADP

ATP

glycolysis rate is increased

glycolysis rate is decreased

Fructose 6-phosphate AMP heat Pi F-1,6-BPase H2O ATP PFK-1 ADP + AMP

Fructose 1,6-biphosphate Futile cycle is considered as a substrate cycle. The combined reactions catalyzed by PFK-1 and F-1,6-BPase result in net ATP hydrolysis: ATP + H2O ADP + Pi + heat

The net reaction shows a useless consumption of ATP. The inhibition of AMP on F-1,6-BPase results to a large increase in the amount of fructose 1,6-biphosphate.

Regulation of PFK-1 PFKby intracellular pH

(a) Glucose transport into the cell. (b) All work performances that convert ATP back to ADP and Pi. (c) latate-H symport

Accumulation of Intracellular Lactic Acid

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Regulation of PFK-1 PFKby intracellular pH

excessive anaerobic glycolysis accumulation of intracellular lactic acid

decrease in blood pH

PFK-1 -

inadequate blood flow

Lactic acidosis

High [H ]

decrease in intracellular pH

What is lactic acidosis?

It is a form of metabolic acidosis caused by the decrease in blood pH and bicarbonate concentration that buffers the acid as a result of elevated blood lactate levels. lactate production lactate utilization tissues that lack O and mitochondria much lactate The major fate of lactate in the body is either complete combustion to CO and H O or conversion back to glucose by gluconeogenesis. less tissues that have O and mitochondria

[blood lactate] is not greater than 1.2 mM

Treatment of lactic acidosis

PHENFORMIN

 a drug that is used to treat the hyperglycemia of type 2 diabetes  induces lactic acidosis as a consequence of inhibition of complex I of the mitochondrial electron transport chain Thiamin deficiency causes lactic acidosis as a consequence of loss of activity of the pyruvate dehydrogenase complex.

THIAMIN

restoration of circulation of oxygenated blood

Malignant Hyperthermia

C
increase in body temperature

metabolic and respiratory acidosis anesthetichalothane exposure hyperkalemia abnormality of ryanodine receptor in sarcoplasmic reticulum muscle rigidity

inappropriate release of Ca from the sarcoplasmic reticulum

uncontrolled stimulation of heatproducing processes

atherosclerosis

spasm narrowing of coronary arteries

formation of blood clot

imbalance between demand for and supply of blood flow to cardiac muscles strangling chest pain associated with reversible myocardial ischemia

persists long

Angina Pectoris

Myocardial Infarction

Treatment of Angina Pectoris and Myocardial Infarction

nitroglycerin and other nitrates

 causes venodilation  lowers the oxygen requirement of the heart  washes out lactic acid  act as coronary vasodilators
 prevents the increase

calcium channel blockers adrenergic blockers coronary bypass operation

in myocardial oxygen consumption

 veins are removed from the

leg and interposed between the aorta and coronary arteries so as to bypass the portion affected by atherosclerosis

Regulation of PFK-1 by Citrate PFK-

fatty acids ketone bodies used as oxidative fuels in replacement of glucose PFK-1 aerobic oxidation -

CITRATE

pyruvate

The decrease in glucose utilization helps preserve glucose for tissues that are absolutely glucose-dependent.

Fructose 2,6-biphosphate 2,6(F-2,6-BP) (F-2,6Potent Allosteric Activator of PFK-1 PFK-

Fructose 2,6-biphosphate is 2,6

an important factor in the livers maintenance of blood glucose non glycolytic metabolite that is a potent allosteric activator of PFK-1 and an inhibitor of F-1,6-Bpase independently discovered in 1980 by Simon Pilkis, by Emile Van Schaftingen and HenriGry Hers, and by Kosaku Uyeda

Production of F-2,6-BP F-2,6-

F-2,6-BP is produced as a side product rather than as an intermediate of glycolysis. Intracellular concentration of F-2,6-BP is dependent on the balance between its rates of synthesis and degradation.
Reactions Involved in the Formation and Degradation of Fructose 2,6-biphosphate
Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Production of F-2,6-BP F-2,6-

PFK-1 produces and intermediate, F-1,6-BP, of glycolysis while PFK-2 produces a positive allosteric effector, F-2,6-BP, of PFK-1. The activities of PFK-2 and F-2,6-BPase reside in the same protein which is a bifunctional enzyme called, 6-phosphofructo-2-kinase/fructose 2,6biphosphatase (PFK-2/F-2,6-Bpase).
Reactions Involved in the Formation and Degradation of Fructose 2,6-biphosphate
Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Structure of the Bifunctional Enzyme

~100 kd homodimer N-terminal (blue) is the PFK-2 domain with 246 aa residues. C-terminal (yellowgreen) is the F-2,6-BPase domain with 213 aa residues. The succinate occupies the presumed F6Pbinding pocket of the PFK-2 domain.
X-ray structure of the H256A mutant of rat testis PFK-2/FBPase-2
Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Structure of the Bifunctional Enzyme

AMPPNP or Adenosine5-(,-imido)triphosphate is a nonhydrolyzable ATP analog

The F6P and Pi marks the active site of F-2,6BPase for F-2,6-BP.
X-ray structure of the H256A mutant of rat testis PFK-2/FBPase-2
Biochemistry, 4th Edition edited by Donald Voet and Judith G. Voet 2011 John Wiley & Sons, Inc.

Hormonal Control of PFK-1 PFKby cAMP and F-2,6-BP F-2,6cells of pancreas

Mechanism by which glucagon inhibits hepatic glycolysis.

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Regulation of Enzymes by PhosphorylationPhosphorylationDephosphorylation

The active enzyme is called the a form while the inactive enzyme is called the b form.

The ratio of phosphorylated to dephosphorylated enzyme depends on the relative activities of PKA and phosphoprotein phosphatase.

General Model for Regulation of Enzymes by Phosphorylation-Dephosphorylation

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Regulation of PFK-2/F-2,6-Bpase PFK-2/F-2,6by Phosphorylation

Phosphorylation of the liver PFK-2/F-2,6-BPase enzyme at its Ser 32 inhibits its PFK-2 activity and activates its F-2,6-BPase activity.
Mechanism for Covalent Modification of PFK-2/F-2,6-Bpase.
Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Phosphorylation at Ser 32

Schematic Diagram of the Primary Structure of the Liver PFK-2/F-2,6-Bpase.

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Mechanism of Glucagon and Epinephrine Inhibition of Hepatic Glycolysis

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

low blood glucose level high blood glucagon levels activation of PKA phosphorylation of PFK-2/F-2,6-BPase decreased concentration of F-2,6-BP inactivation of PFK-2 moiety activation of F-2,6-BPase moiety

Gluconeogenesis is favored

Tyr kinase activity

Mechanism for Acceleration of Hepatic Glycolysis by Insulin

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Phosphorylatio n of the bifunctional enzyme in the heart increases the concentration of F-2,6-BP.

Mechanism for Acceleration of Glycolysis in the Heart in Response to Epinephrine

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

The isoenzyme of the bifunctional enzyme expressed in the heart is different from the isoenzyme expressed in the liver. The kinase moiety is activated while the phosphatase moiety is inhibited.
Schematic Diagram of the Primary Structure of the Heart PFK-2/F-2,6-Bpase
Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.

Key Points
1. Glucagon and epinephrine are extracellular signals that stop liver from using glucose. 2. Fructose 2,6-biphosphate is an intracellular signal that promotes glucose utilization by the tissue. 3. Insulin acts to stimulate the rate of glycolysis. 4. Epinephrine inhibits glycolysis in the liver to conserve glucose for use by other tissues. 5. Epinephrine stimulates glycolysis in the heart to meet the increased need for ATP.

#2

Pyruvate Kinase (PK)

A Regulatory Enzyme of Glycolysis

Physiological concentration of ATP greatly inhibits the activity of pyruvate kinase. Fructose 1,6-biphosphate greatly activates the activity of pyruvate kinase.

Covalent Modification in PKA Inhibits Pyruvate Kinase

Textbook in Biochemistry, 7th Edition edited by Thomas M. Devlin 2011 John Wiley & Sons, Inc.