Download Glucose Metabolism Glycolysis Expectations

3/1/2017
Glucose Metabolism
Pratt and Cornely, Chapter 13
Glycolysis Expectations
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Memorize/learn Figure 13.2
Know overall reaction and stages
Explain chemical logic of each step
Enzyme mechanisms presented in book
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Glycolysis
• Ten enzymes that take glucose to pyruvate
• Cytosol
• ATP and NADH
Reactions and Enzymes of Glycolysis
ATP
ATP
Pi + NAD+
ADP
2x
ADP
ADP
NADH
ADP
2x
2x
ATP
2x
ATP
• Hexose and triose phases
• Energy input and payoff phases
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Energy Input
Energy Payoff
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Know...
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Substrates Co‐substrates Products Enzyme names
1. Hexokinase • Previous concepts: Induced fit, kinase
• Utilizes 1 ATP
• Chemical logic?
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Problem 3
• (Notice miswording) The Go’ value for hexokinase is ‐16.7 kJ/mol, but it is twice as spontaneous under cellular conditions.
– What is the ratio of G‐6‐P to glucose under cellular conditions when the ratio of ATP:ADP is 10:1?
– How high would the ratio of G‐6‐P to glucose have to be to reverse the hexokinase reaction by mass action?
2. Phosphoglucose Isomerase
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Previous concepts: Isomerization
CONCEPT: Near‐equilibrium
Chemical logic:
Stereochemistry—reverse does not produce mannose!
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3. PFK‐1
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Previous concepts: Allosteric inhibition
Utilizes 1 ATP
Pathway logic:
First committed step of glycolysis
– Why?
– regulation
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Regulation: Bacteria vs. Human
4. Aldolase
• Previous concepts: Standard free energy is +23kJ, but it is a near equilibrium reaction
• Pathway logic:
• Beginning of triose stage
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Aldolase
Mechanism
5. Triose Phosphate Isomerase
• Previous concepts: Catalytic perfection
• Pathway logic:
• Most similar to which previous reaction?
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6. Glyceraldehyde‐3‐P DH
• Previous concepts: Redox and dehydrogenase
• Pathway logic: Utilizes negative free energy of _____________ to drive nonspontaneous formation of ___________________
GAPDH Mechanism
Be able to draw mechanism with full NAD+ structure
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7. Phosphoglycerate Kinase •
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Previous concepts: High energy bond
Produces 2 ATP: substrate level phosphorylation
Pathway logic?
Coupled to reaction 6 by Le Chatelier
Coupled Reactions
• GAPDH = 6.7 kJ/mol
• PG Kinase = ‐18.8 kJ/mol
• Overall: A set of near‐equilibrium reactions at cellular concentration 10
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8. Phosphoglycerate Mutase
• Previous concepts: Covalent catalysis
• Pathway logic:
• Mutase—isomerization with P transfer
Mechanism
• Not a simple transfer
• What happens if the bisphosphate escapes?
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9. Enolase
• Concept: Phosphoryl group transfer potential
• Chemical logic?
10. Pyruvate Kinase
• Production of 2 ATP
• VERY high energy bond allows formation of _________ while still being irreversible
• Regulation: F‐1,6‐BP can act as a feed‐forward activator to ensure fast glycolysis
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Overall Energetics
• Standard Free energies are up and down
• Free energies under cellular conditions are downhill or near zero
– Three irreversible
Fate of Pyruvate
Amino acid
and nitrogen
metabolism
Gluconeogenesis
Aerobic Energy
Anaerobic in
higher organisms
Anaerobic in
microorganisms
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The Problem of Anaerobic Metabolism
• With oxygen, the NADH produced in glycolysis is re‐oxidized back to NAD+
• NAD+/NADH is a co‐substrate which means…
• If there is no oxygen, glycolysis will stop because…
• The solution to the problem is to…
The solution in Yeast
• Pyruvate is decarboxylated to acetaldehyde
• Acetaldehyde transformed to ethanol – What type of reaction?
– What cofactor?
• NAD+ is regenerated to be reused in GAPDH
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The Solution in Us
• Lactate formation: Draw a mechanism
• Balanced equation
We don’t operate anaerobically...
• Most energy still trapped in lactate
• Back to pyruvate, then acetyl‐CoA
• Citric acid cycle
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Other sugars enter glycolysis
High fructose diet puts sugars through glycolysis while avoiding major regulation step
Glucose Metabolism Overview
• Keep the main pathway purposes distinct
• But learn details of chemistry and regulation based on similarities
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Glucose Metabolism Overview
Energy Production
ATP
Pentose
Phosphate
Pathway
Ribose,
NADPH
Pyruvate
• Gluconeogenesis
• Glycogen metabolism
• Pentose Phosphate Pathway
Glycogen Synthesis
DHAP
Glycogen
OH
O
HO
HO
OH
OH (P)
Glycerol
(Triacylglycerides)
DHAP
Glycogen
Lactate
Glycogen Degradation
Pyruvate
Amino Acids
Gluconeogenesis
Precursors for Gluconeogenesis
OH
• Names of compounds?
• Type of reaction?
• Type of enzyme?
• Cofactor(s)?
OH
OH
O
OH
OPO3
O
O
O
O
NH2
O
O
O
O
O
OH
O
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Chemistry of Gluconeogenesis
• Pyruvate transformed to glucose
• But energetically costly—no perpetual motion machine!
• Points of regulation
Glycolysis
• Gluconeogenesis
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• Step 10: no ATP back
• Step 8: no ATP back
• Step 3: costs 2 ATP
• Step 1a/b: costs 4 ATP equivalents
Step 1: costs 1 ATP
Step 3: costs 1 ATP
Step 7: makes 2 ATP
Step 10: makes 2 ATP
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Step 1a
• Pyruvate Carboxylase
– Biotin
– Costs ATP to make driving force for next reaction
• TIMES TWO!
– First step in biosynthesis of glucose and many other molecules
• Related to which amino acid?
Mechanism
• Mixed anhydride activates carboxyl group
• Coupled through biotin coenzyme
• Enolate attacks activated carboxylate
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Step 1b
• PEP carboxykinase
– ATP cost to restore PEP
• TIMES TWO!
– CO2 loss drives rxn
Step 8
• Fructose‐1,6‐bisphosphatase
• No additional energy input
• Phosphate ester hydrolysis is spontaneous
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Step 10
• Glucose 6‐phosphatase
– Liver (and others)
– Not in muscle
Problem 34
• A liver biopsy of a four‐year old boy indicated that the F‐1,6‐Bpase enzyme activity was 20% normal. The patient’s blood glucose levels were normal at the beginning of a fast, but then decreased suddenly. Pyruvate and alanine concentrations were also elevated, as was the glyceraldehyde/DHAP ratio. Explain the reason for these symptoms. 21
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Key Regulation
• At the committed step in glucogenic cells
• Principle of Reciprocal Regulation
• Local regulation vs Hormone regulation
Key Regulation
• Local regulation
– AMP/ATP (energy charge)
– Citrate (feedback)
• Hormone regulation
– Fructose‐2,6‐bisphosphate
• Gluconeogenesis is inhibited
• Glycolysis is stimulated
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Problem 39
• Brazilin, a compound found in aqueous extracts of sappan wood, has been used to treat diabetics in Korea. It increases the activity of the enzyme that products F‐2,6‐BP and stimulates the activity of pyruvate kinase. What is the effect of adding brazilin to liver cells in culture? Why would brazilin be an effective treatment for diabetes?
Glucose Metabolism Overview
Energy Production
ATP
Pentose
Phosphate
Pathway
Ribose,
NADPH
Pyruvate
• Gluconeogenesis
• Glycogen metabolism
• Pentose Phosphate Pathway
Glycogen Synthesis
DHAP
Glycogen
OH
O
HO
HO
OH
OH (P)
Glycerol
(Triacylglycerides)
DHAP
Glycogen
Lactate
Glycogen Degradation
Pyruvate
Amino Acids
Gluconeogenesis
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Glycogen
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Storage molecule
Primer necessary
Very large!
Multiple ends allow for quick synthesis and degradation
Chemistry of Synthesis
• Step 1
• Near equilibrium
• The link to glucose‐6‐phophate, our central molecule
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Chemistry of Synthesis
• Step 2
• Count high energy bonds
• Pyrophosphatase
– Common motiff
• UDP‐glucose: activated for incorporation
Chemistry of Synthesis
• Step 3
• Glycogen synthase
• Growing end is non‐reducing
• UDP released
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Branching
Energetics of Synthesis
• Total cost: one ATP equivalent from G‐6‐P
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Four Degradation Enzymes
• Two enzyme debranching process
• Glycogen phosphorylase
• phophoglucomutase
Chemistry of Degradation
• Glycogen phosphorylase
• Key Regulation site
• Inorganic phosphate as a nucleophile
• Remake G‐1‐P with no ATP cost
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Debranching
• Transfer branch
• (16)glycosidase
Overall Energetics
OH
O
HO
Glucose-6-P
HO
OH
2 Pi
O
P-P-Uridine
UDP
UTP
OH
OH
O
HO
OH O
HO
HO
OH
O
HO
P
O
HO
O
OH
O
O
O
Pi
OH
O
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Key Regulatory Enzymes
OH
O
Glycogen Synthase
HO
Glucose-6-P
HO
OH
2 Pi
O
P-P-Uridine
UDP
UTP
OH
OH
O
HO
OH O
HO
HO
OH
O
HO
P
O
HO
O
OH
O
O
O
Pi
Glycogen Phosphorylase
OH
O
Glycogen Storage Diseases
Many disrupt glycogen breakdown in muscle and/or liver
(hypoglycemia, enlarged liver, muscle cramps...)
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Glucose Metabolism Overview
Energy Production
ATP
Pentose
Phosphate
Pathway
Ribose,
NADPH
Pyruvate
• Gluconeogenesis
• Glycogen metabolism
• Pentose Phosphate Pathway
Glycogen Synthesis
DHAP
Glycogen
OH
O
HO
HO
OH
OH (P)
Glycerol
(Triacylglycerides)
DHAP
Glycogen
Lactate
Glycogen Degradation
Pyruvate
Amino Acids
Gluconeogenesis
Pentose Phosphate Pathway
• Dual Purpose
– Synthesis of “reducing potential”
– Synthesis of 5‐carbon sugars
• At cost of one carbon worth of carbohydrate
• Net reaction:
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Physiological Purposes
Glutathione: Protection from Oxidation
• Glutathione is the redox buffer of the cell
• Regenerated by NADPH
• PPP especially important in RBC because it is only means to generate reducing power (no mitochondria)
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Complex, 2‐Stage Process
• Oxidative Stage
– Generates reducing power and ribose
• Non‐oxidative stage
– Regenerates 3‐ and 6‐carbon sugars from 5 carbon sugars
Oxidative Stage Step 1:
• G‐6‐P DH
• Lactone formation
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Oxidative Stage Step 2:
• Also a spontaneous hydrolysis
• Practice mechanism, carbohydrate orientation
Oxidative Stage Step 3:
• Oxidative decarboxylation
– Which is first, oxidation or decarboxylation?
• We will see this process again
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Biosynthesis of Ribose
Non‐oxidative Stage
• To understand purpose, realize that we generally need to make much more NADPH than ribose
• Problem: stuck with C5, but need C6 and C3
• Solution: “Shunt” C5 back to C6 through near‐equilibrium reactions
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PPP Reactions
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Epimerase
Isomerase
Transketolase
Transaldolase
Epimerase
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Transketolase
• Use cofactor (B1) to overcome chemical problem
Mechanism
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Transketalase, Transaldolase
• Transketalase
– Transfer of an acyl anion
– Requires TPP to stabilize unstable anion
• Transaldolase
– Transfer of somewhat stable enolate ion
– Schiff base helps
• Be able to recognize need for TK vs. TA
Transketalase or transaldolase?
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Different Modes for Different Purposes
Problem 58
• A given metabolite may follow more than one metabolic pathway. List all possible fates of glucose‐6‐P in (a) a liver cell and (b) a muscle cell.
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Summary
of glucose
metabolism
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