Carb Metabolism Regulation: Glycolysis, Gluconeogenesis, and Glycogen, Exercises of Enzymes and Metabolism

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BI/CH 422/

ANABOLISM OUTLINE:

Overview of Photosynthesis Key experiments: Light causes oxygen, which is from water splitting (Hill) NADPH made (Ochoa) Separate from carbohydrate biosynthesis (Rubin & Kamen) Light Reactions energy in a photon pigments HOW Light absorbing complexes Reaction center Photosystems (PS) PSI – oxygen from water splitting PSII – NADPH Proton Motive Force – ATP Overview of light reactions Carbon Assimilation – Calvin Cycle Stage One – Rubisco Carboxylase Oxygenase Glycolate cycle Stage Two – making sugar Stage Three - remaking Ru 1,5P 2 Overview and regulation Calvin cycle connections to biosyn. C4 versus C3 plants Kornberg cycle - glyoxylate

Carbohydrate Biosynthesis in Animals

precursors Cori cycle Gluconeogenesis reversible steps irreversible steps – four energetics 2-steps to PEP mitochondria Pyr carboxylase-biotin PEPCK FBPase G6Pase Glycogen Synthesis UDP-Glc Glycogen synthase Branching Pentose-Phosphate Pathway Oxidative phase Non-oxidative/recycling phase ROS and NADH/NADPH shuttles

Regulation of Carbohydrate Metabolism

Acetyl-CoA/Pyruvate Pyruvate/PEP F6P/FBP: Fru 2,6P 2 Glc/Glc6P Glycogen

Anaplerotic reactions

Regulation of

Carbohydrate

Metabolism

Catabolism vs. Anabolism

• Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme.
• Also where there are junctions.
• Can you name those enzymes?

Regulation of Carbohydrate

Metabolism

Glycolysis versus Gluconeogenesis

Glycogenolysis versus Glycogen Synthesis

Pyruvate Dehydrogenase Complex

Catabolism Anabolism

Pyruvate Kinase – PEPCK

• Pyruvate carboxylase

Phosphofructo- kinase-

Fructose 1,6- bisphosphatase

-

Hexokinase Glucose 6- phosphatase

Phosphorylase Glycogen Synthase

Glc 1-P

Glycogen

UDP-Glc

GlycogenPhosphorylase Glycogen Synthase

phosphoglucomutase

Acetyl-CoA

UDP-glucosepyrophosphorylase

Gene controlled

Catabolism vs. Anabolism

✓

Regulation of Pyruvate

Kinase

• Allosterically activated by

fructose-1,6-bisphosphate

• increase flow through glycolysis
• Feed-forward activation
• Allosterically inhibited by

signs of abundant energy

supply.

Regulation of Carbohydrate

Metabolism

• ATP
• acetyl-CoA and long-chain fatty acids
• alanine (enough amino acids)

Inactivated by phosphorylation in response to signs

of glucose depletion (low blood-

glucoseàglucagon) (liver only)

Glucose from liver is exported to the brain and other vital organs.

All tissues

Liver only

This is not the only time we’ll see hormonal control of these pathways.

(under hormonal control)

Regulation of Pyruvate

Kinase

• Allosterically activated by

fructose-1,6-bisphosphate

• increase flow through glycolysis
• Feed-forward activation
• Allosterically inhibited by

signs of abundant energy

supply.

Metabolism

• ATP
• acetyl-CoA and long-chain fatty acids
• alanine (enough amino acids)

Inactivated by phosphorylation in response to signs

of glucose depletion (low blood-

glucoseàglucagon) (liver only)

Glucose from liver is exported to the brain and other vital organs.

All tissues

Liver only

This is not the only time we’ll see hormonal control of these pathways.

(under hormonal control)

Regulation of Pyruvate

Carboxylase

• Allosteric activation of pyruvate

carboxylase by Acetyl-CoA

• stimulates glucose synthesis via gluconeogenesis because plenty of acetyl-CoA signals plenty of CAC intermediates
• Notice the reciprocal control of PDH

Complex by acetyl-CoA

Regulation of Carbohydrate

Metabolism

• Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme.
• Can you name those enzymes?

Metabolism

Glycolysis versus Gluconeogenesis

Glycogenolysis versus Glycogen Synthesis

Pyruvate Dehydrogenase Complex

Pyruvate Kinase – PEPCK

• Pyruvate carboxylase

Phosphofructo- kinase-

Fructose 1,6- bisphosphatase

Hexokinase Glucose 6- phosphatase

Phosphorylase

Glc 1-P

Glycogen

UDP-Glc

GlycogenPhosphorylase Glycogen Synthase

phosphoglucomutase

Acetyl-CoA

UDP-glucosepyrophosphorylase

Glycogen Synthase

Catabolism Anabolism

Regulation of Carbohydrate

Metabolism

Regulation of

Phosphofructokinase-

versus

Fructose 1,6-bisphosphatase-

• Fructose-6-phosphate à fructose 1,6-bisphosphate is the commitment step in glycolysis.
• While ATP is a substrate, ATP is also a negative effector.
  • Do not spend glucose in glycolysis if there is plenty of ATP.
  • Same for citrate, if there is plenty of citrate, do not waste glucose
• Low energy charge inhibits biosynthesis of Glc.
  • Go glycolysis if AMP is high and ATP is low.
  • Go gluconeogenesis if AMP is low.

Homeostatic level of Fru 6-P

Is this a typo? Bumble bees are missing an FBPasethat responds to AMP

High [AMP]

Low [AMP]

Metabolism

Regulation of

Phosphofructokinase-

versus

Fructose 1,6-bisphosphatase-

• Fructose-6-phosphate à fructose 1,6-bisphosphate is the commitment step in glycolysis.
• While ATP is a substrate, ATP is also a negative effector.
  • Do not spend glucose in glycolysis if there is plenty of ATP.
  • Same for citrate, if there is plenty of citrate, do not waste glucose
• Low energy charge inhibits biosynthesis of Glc.
  • Go glycolysis if AMP is high and ATP is low.
  • Go gluconeogenesis if AMP is low.

Homeostatic level of Fru 6-P

Is this a typo? Bumble bees are missing an FBPasethat responds to AMP

High [AMP]

Low [AMP]

Fructose 2,6- (bis) phosphate

( b D-Fru-2,6P 2 )

•NOT a glycolytic intermediate, only a regulator

•Produced specifically to regulate glycolysis and gluconeogenesis

• activates phosphofructokinase-1(PFK-1) (glycolysis)
• inhibits fructose 1,6-bisphosphatase (FBPase-1) (gluconeogenesis)

Regulation of Carbohydrate

Metabolism

Enzyme for synthesis and degradation of Fru 2,6P 2 done with a dual-function enzyme: PFK-2/FBPase-2*

b D-Fru-1,6P 2

*6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Regulation of Glycolysis and

Gluconeogenesis by Fru-2,6P (^2)

Metabolism

• With Fru 2,6P 2 (130 nM), Go glycolysis.
• With Fru 2,6P 2 (1300 nM), Stop gluconeogenesis.
• Without Fru 2,6P 2 , STOP glycolysis GO gluconeogenesis.

What controls PFK-2/FBPase-2?

Regulation of Fru-2,6-P 2 Levels

Regulation of Carbohydrate

Metabolism

Glucagon

Drop relieves the activation of PFK-1, effectivelyinhibiting glycolysis. Drop increases activity ofFBPase-1, stim ulating gluconeogenesis

Insulin

Structurally, this enzyme, with its two activities is different than those in glycolysis and gluconeogenesis (i.e., they are conjoined, rather than independent) and are regulated via phosphorylation.

Regulation of Fru-2,6-P 2 Levels

Metabolism

Glucagon

Drop relieves the activation of PFK-1, effectivelyinhibiting glycolysis. Drop increases activity ofFBPase-1, stim ulating gluconeogenesis

Insulin

Structurally, this enzyme, with its two activities is different than those in glycolysis and gluconeogenesis (i.e., they are conjoined, rather than independent) and are regulated via phosphorylation.

• Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme.
• Can you name those enzymes?

Regulation of Carbohydrate

Metabolism

Glycolysis versus Gluconeogenesis

Glycogenolysis versus Glycogen Synthesis

Pyruvate Dehydrogenase Complex

Pyruvate Kinase – PEPCK

• Pyruvate carboxylase

Phosphofructo- kinase-

Fructose 1,6- bisphosphatase

Hexokinase Glucose 6- phosphatase

Phosphorylase

Glc 1-P

Glycogen

UDP-Glc

GlycogenPhosphorylase Glycogen Synthase

phosphoglucomutase

Acetyl-CoA

UDP-glucosepyrophosphorylase

Glycogen Synthase

Catabolism Anabolism

There Are Four Isozymes

of Hexokinase (I-IV)

•Isozymes are different enzymes that catalyze the

same reaction.

–typically share similar sequences –may have different kinetic properties –can be regulated differently

•HK I is expressed in all tissues, to different levels.

•HK IV (glucokinase) is only expressed in the liver

and pancreas.

Metabolism

Regulation of

Hexokinase

HK-I versus HK-IV

Glucokinase Is Regulated by Sequestration

⊕

Fructose 1-phosphate

–has higher K m , so responsive to higher [glucose] –not inhibited by glucose-6-phosphate, so can function at higher [glucose] –functions to clear blood glucose at higher [glucose] for storage as glycogen –Glc activates release/Fru inhibits

• Regulated enzymes often correspond to points in the pathways that have the same substrate and product, but a different enzyme.
• Can you name those enzymes?

Regulation of Carbohydrate

Metabolism

Glycolysis versus Gluconeogenesis

Glycogenolysis versus Glycogen Synthesis

Pyruvate Dehydrogenase Complex

Pyruvate Kinase – PEPCK

• Pyruvate carboxylase

Phosphofructo- kinase-

Fructose 1,6- bisphosphatase

Hexokinase Glucose 6- phosphatase

Phosphorylase Glycogen Synthase

Glc 1-P

Glycogen

UDP-Glc

GlycogenPhosphorylase Glycogen Synthase

phosphoglucomutase

Acetyl-CoA

UDP-glucosepyrophosphorylase

Catabolism Anabolism

AMP AMP

•Glycogen phosphorylase cleaves glucose

residues off glycogen, generating glucose-1-

phosphate (Glc 1P).

• Phosphorylation activates glycogen phosphorylase- b –Phosphorylase- b Kinase –Accentuated by allosteric binding of AMP (muscle only)
• Dephosphorylation inhibits glycogen phosphorylase- a –Phosphoprotein phosphatase-1 (PP1) –Accentuated by allosteric binding of Glc (in liver only)

Nobel Prize 1972 Earl Sutherland 1915-

Glc Glc

Regulation of

Glycogen Phosphorylase

Metabolism

AMP AMP

•Glycogen phosphorylase cleaves glucose

residues off glycogen, generating glucose-1-

phosphate (Glc 1P).

• Phosphorylation activates glycogen phosphorylase- b –Phosphorylase- b Kinase –Accentuated by allosteric binding of AMP (muscle only)
• Dephosphorylation inhibits glycogen phosphorylase- a –Phosphoprotein phosphatase-1 (PP1) –Accentuated by allosteric binding of Glc (in liver only)

Nobel Prize 1972 Earl Sutherland 1915-

Glc Glc

Regulation of

Glycogen Phosphorylase

Regulation of Carbohydrate

Metabolism

Metabolism

•Glycogen synthase adds glucose residues to

glycogen using UDP-Glc.

• Phosphorylation inhibits glycogen synthase- a –Its complicated, responding to multiple signals –Example: First Casein Kinase-2 (CKII), then Glycogen Synthase Kinase-3 (GSK3)

Regulation of glycogen synthase/glycogen phosphorylase is somewhat similar to regulation of PDH complex

Regulation of

Glycogen

Synthase

• Dephosphorylation activates glycogen synthase- b –Phosphoprotein phosphatase-1 (PP1) (in liver it’s a different PP) –PP1 is bound to GS-b
• Also, feedforward control by glucose and Glc-6P –Binding causes a conformation favorable for PP-1 binding –Binding does not allow GSK-3 access to phosphorylation sites

Regulation of Carbohydrate

Metabolism

•Glycogen synthase adds glucose residues to

glycogen using UDP-Glc.

• Phosphorylation inhibits glycogen synthase- a –Its complicated, responding to multiple signals –Example: First Casein Kinase-2 (CKII), then Glycogen Synthase Kinase-3 (GSK3)

Regulation of glycogen synthase/glycogen phosphorylase is somewhat similar to regulation of PDH complex

Regulation of

Glycogen

Synthase

• Dephosphorylation activates glycogen synthase- b –Phosphoprotein phosphatase-1 (PP1) (in liver it’s a different PP) –PP1 is bound to GS-b
• Also, feedforward control by glucose and Glc-6P –Binding causes a conformation favorable for PP-1 binding –Binding does not allow GSK-3 access to phosphorylation sites

Metabolism

•Glucagon/epinephrine signaling pathway

activated when there is a NEED for energy

• starts phosphorylation cascade via cAMP
• cAMP activates PKA
• PKA activates phosphorylase-b kinase
• this kinase activates glycogen phosphorylase
• Massive degradation of glycogen
  • In muscle Glc1PàGlc6Pàglycolysis
  • In liver Glc1PàGlc6PàGlc

Nobel Prize 1992 Edwin Krebs 1918-

Glycogen Phosphorylase Cascade

& Hepatocyte b -adenoreceptor glucagon receptor G-proteinactivation

• There is reciprocal inhibition of GS
  • PKA phosphorylates G (^) M , which is bound to PP1 on GS, thus dissociating it.
  • PKA also phosphorylates PP1-inhibitor protein, which binds and inactivates the free PP1, thus leaving GS-℗ and inactive

Anaplerotic Reactions

• We introduced the citric acid cycle as a key catabolic pathway.
• It has an equal, if not more important, role in anabolism.
  • The biosynthesis of biological precursors has to begin with elementary materials
  • Many, if not most, of these starting points come from the several intermediates in the Kreb’s cycle.
  • This was first appreciated by Hans Kornberg: how to organisms grow on carbohydrates only? o The term comes from the Greek, to ”fill up” or replenish o Recall that without this replenishment, the TCA cycle would grind to a halt o Anaplerotic reactions are critical

Enzyme Kornberg discovered

Anabolism

Catabolism

(PEPCK)

Kornberg Cycle = Glyoxylate Cycle

Anaplerotic Reactions

2 Acetyl-CoA + NAD +^ ⇌^ Succinate + 2 CoASH + NADH

• Was intrigued by the fact that bacteria could grow very effectively on a little ammonium and phosphate salts with acetate (2C)
• From these they can synthesize all the components of the cell; DNA, DNA, proteins, membrane lipids, cytochromes, everything…..
• How do you build all this from a 2-carbon compound knowing how the Kreb’s cycle works?

Dr. Kornberg: Lecture 03.29.17 (31:32- 35:09) 3.5 min

Anaplerotic Reactions

• Purine Nucleotide Cycle
  • was first thought be be part of nucleotide degradation or synthesis
  • In muscle, its now realized as an important anaplerotic pathway

H 2 O + Aspartate + GTP ⇌ NH 4 +^ + GDP + P (^) i + Fumarate

Myoadenylate deaminase deficiency Loss of AMP deaminase-1, the muscle-specific isozyme Causes exercise-induced muscle pain

Anaplerotic Reactions

• Intermediates in the citric acid cycle

can be used in biosynthetic pathways.

• Must replenish the intermediates in

order for the cycle and central

metabolic pathway to continue.

• In animals, these 4-carbon

intermediates are formed by

carboxylation of 3-carbon precursors.

Pyruvate carboxylase deficiency

• an inherited metabolic disorder where anaplerosis is greatly reduced.
• What is the problem?
• How to treat this disorder?
• Other anaplerotic substrates such as the odd-carbon-containing triglyceride triheptanoin are used

Aspartate + GTP NH 4 +^ + GDP + Pi + Fumarate Muscle

AMP deaminase, adenylosuccinate synthetase, adenylosuccinate lyase

Enzyme Kornberg discovered

• Gluconeogenesis, a process by which cells can use a variety of metabolites for the synthesis of glucose
• The differences between glycolysis and gluconeogenesis
  • how they are both made thermodynamically favorable
  • how they are differentially regulated to avoid a futile cycle
• The pentose phosphate pathway, a process by which cells can generate pentose phosphates and NADPH. The pentose phosphates can be regenerated into glucose 6-phosphate, for which NO ATP is required.
• living organisms regulate the flux of metabolites through metabolic pathways by:
  • increasing or decreasing enzyme concentrations
  • activating or inactivating key enzymes in the pathway
• the activity of key enzymes in glycolysis and gluconeogenesis is tightly and coordinately regulated via various activating and inhibiting metabolites (Fru 2 ,6P 2 )
• glycogen synthesis and degradation is regulated by hormones insulin, epinephrine, and glucagon that report on the levels of glucose in the body
• the citric acid cycle plays important anabolic roles in the cell: Anaplerosis
• organisms have multiple ways to replenish intermediates that are used in other pathways: Lipid and Nitrogen biosynthesis….......

ANABOLISM I: Summary

What we learned: