Transcript File

Step 8: Migration of the Phosphate
Step 8: Migration of the Phosphate
• Rationale:
– Be able to form high-energy phosphate compound
• Mutases catalyze the (apparent) migration of functional
groups
– One of the active site histidines is post-translationally modified
to phosphohistidine
– Phosphohistidine donates its phosphate to O2 before retrieving
another phosphate from O3
• 2,3-bisphosphoglycerate intermediate
• Note that the phosphate from the substrate ends up bound
to the enzyme at the end of the reaction
Step 8: Migration of the Phosphate
• Thermodynamically unfavorable/reversible
– Reactant concentration kept high by PGK to push forward
Mechanism of Phosphoglycerate Mutase:
Base Catalyzed Phosphoryl Transfer
Mechanism of Phosphoglycerate Mutase:
Acid Catalyzed Phosphoryl Transfer
Step 9: Dehydration of 2-PG to PEP
Step 9: Dehydration of 2-PG to PEP
• Rationale
– Generate a high-energy phosphate compound
• 2-Phosphoglycerate is not a good enough phosphate
donor
– Two negative charges in 2-PG are fairly close
– But loss of phosphate from 2-PG would give a secondary alcohol
with no further stabilization
• Slightly thermodynamically unfavorable/reversible
– Product concentration kept low to pull forward
Step 10: 2nd Production of ATP
Step 10: 2nd Production of ATP
• Rationale
– Substrate-level phosphorylation to make ATP
– Net production of 2 ATP/glucose
• Loss of phosphate from PEP yields an enol that
tautomerizes into ketone
– Tautomerization
• effectively lowers the concentration of the reaction
product
• drives the reaction toward ATP formation
Pyruvate Tautomerization
Drives ATP Production
Step 10: 2nd Production of ATP
• Pyruvate kinase requires divalent metals (Mg2+ or Mn2+)
for activity
• Highly thermodynamically favorable/irreversible
– Regulated by ATP, divalent metals, and other metabolites
4C. Summary of Glycolysis
Glucose + 2 NAD+ + 2 ADP + 2 Pi  2 Pyruvate + 2 NADH + 2 H+ + 2 ATP
• Used:
– 1 glucose; 2 ATP; 2 NAD+
• Made:
– 2 pyruvate
• Various different fates
– 4 ATP
• Used for energy-requiring processes within the cell
– 2 NADH
• Must be reoxidized to NAD+ in order for glycolysis to continue
• Glycolysis is heavily regulated
– Ensure proper use of nutrients
– Ensure production of ATP only when needed
4D. Fates of Pyruvate
5. Glycolysis occurs at elevated rates
in tumor cells
Tumor cells have a higher requirement for glucose due to
a lower efficiency in energy production from glycolysis.
• Complete oxidation of CO2 in healthy cells under aerobic
conditions yields ~30 ATP per glucose.
• Anaerobic metabolism of glucose in tumor cells yields 2
ATP per glucose.
– Glucose transporters and most glycolytic enzymes are
overexpressed in tumors versus normal cells.
– Inhibitors of glycolytic pathways could be effective anticancer
drugs.
Gluconeogenesis
15
1. The Body’s Glucose Need
In mammals, some tissues depend almost completely on
glucose for their metabolic energy. Glucose from the
blood is the sole or major fuel source for:
• Human brain and nervous system
- Brain requires 120 g/day, more than half that is stored as
glycogen in muscles and liver.
• Erythrocytes
• Testes
• Renal medulla
• Embryonic tissues
A mechanism for our bodies to produce glucose is crucial.
2. Gluconeogenesis: Precursors for
Carbohydrates
Notice that mammals cannot
convert fatty acids to sugars.
2. Gluconeogenesis: Precursors for
Carbohydrates
• Animals can produce glucose from sugars or
proteins
– Sugars: pyruvate, lactate, or oxaloacetate
– Protein: from amino acids that can be converted to citric
acid cycle intermediates (or glucogenic amino acids)
• Animals cannot produce glucose from fatty acids
– Product of fatty acid degradation is acetyl-CoA
– Cannot have a net conversion of acetyl-CoA to
oxaloacetate
• Plants, yeast, and many bacteria can do this, thus
producing glucose from fatty acids
3. Glycolysis vs. Gluconeogenesis
Glycolysis occurs mainly
in the muscle and brain.
Gluconeogenesis occurs
mainly in the liver.
3. Glycolysis vs. Gluconeogenesis
• Opposing pathways that are both thermodynamically
favorable
– Operate in opposite direction
• end product of one is the starting compound of the
other
• Reversible reactions are used by both pathways
• Irreversible reaction of glycolysis must be bypassed
in gluconeogenesis
– Highly thermodynamically favorable, and regulated
– Different enzymes in the different pathways
– Differentially regulated to prevent a futile cycle
4. Three bypass reactions of
gluconeogenesis.
A. Conversion of pyruvate to phosphoenolpyruvate
B. Conversion of fructose 1,6-bisphosphate to fructose-6phosphate
C. Conversion of glucose-6-phosphate to glucose
These bypasses are irreversible steps.
4A. Pyruvate to Phosphoenolpyruvate
• Requires two energy-consuming steps
• First step, pyruvate carboxylase converts pyruvate
to oxaloacetate
– Carboxylation using a biotin cofactor
– Requires transport out of the mitochondria via malate
• Second step, phosphoenolpyruvate carboxykinase
converts oxaloacetate to phosphoenolpyruvate
– Phosphorylation from GTP and decarboxylation
– Occurs in mitochondria or cytosol depending on the
organism
4AIa. Synthesis of Oxaloacetate
 This reaction
occurs in the
mitochondria.
Biotin in a cofactor
of the enzyme.
4AIb. Biotin is a CO2 Carrier
4AIc. Oxaloacetate conversion to
malate
 Oxaloacetate has to
be converted to
malate for it to be
transported out of
the mitochondrion.
Mitochondrion
Cytosol
4AIIa. Oxaloacetate to
Phosphoenolpyruvate
Summary of first bypass reaction.
Pyruvate + ATP + GTP + HCO3- →
PEP + ADP + GDP + Pi + CO2
ΔG’°= 0.9 kJ/mol
The reaction is irreversible due to the ready consumption of PEP,
decreasing the amount of product.
4AIII. From Pyruvate to Phosphoenolpyruvate
Isozymes: Two distinct
enzymes that catalyze the
same reaction. They can
have different cellular
locations or metabolic
roles.
4B. Second bypass reaction
Fructose-1-6bisphosphatase
+ Mg2+
+ H2O
+ Pi
ΔG’°= -16.3 kJ/mol
4C. Thirds bypass reaction
Glucose-6-phosphatase
Glucose-6-phosphate + Mg2+
+ H2O
Glucose
+ Pi
ΔG’°= -13.8 kJ/mol
5. Gluconeogenesis is expensive
2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 4 H2O 
Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+
• Costs 4 ATP, 2 GTP, and 2 NADH but physiologically
necessary
– Brain, nervous system, and red blood cells
generate ATP ONLY from glucose
Pentose Phosphate Pathway
33
1. Pentose Phosphate Pathway
1. Pentose Phosphate Pathway
• The main products are NADPH and ribose 5-phosphate
• NADPH is an electron donor
– Reductive biosynthesis of fatty acids and steroids
– Repair of oxidative damage
• For certain tissue, a reducing atmosphere (high ratio of
NADPH to NADP+ and a high ratio of reduced to oxidized
glutathione) helps combat damage by reactive oxygen
species.
• Ribose-5-phosphate is a biosynthetic precursor of
nucleotides
– Used in DNA and RNA synthesis
– Or synthesis of some coenzymes
2. NADPH regulates partitioning into
glycolysis vs. pentose phosphate pathway