Download 1 Lecture 27: Metabolic Pathways Part I: Glycolysis

Biochemistry I
Lecture 27
Nov 7, 2005
Lecture 27: Metabolic Pathways Part I: Glycolysis
Reading in Campbell: Chapter 17.2-17.5
Metabolic pathways are:
What you need to know:
1. Conserved in different organisms.
2. Compartmentalized in Eukaryotes
3. Overall Irreversible (but most of the
individual steps are not)
4. Usually committed after the first step
5. Regulated (usually at first step)
1.
2.
3.
4.
5.
6.
Input and Output metabolites
Steps that control flux
How flux is controlled
Cellular location of metabolic Steps
Selected Enzyme Mechanisms
Selected substrates/products
Central Pathways of Energy Production:
Intracellular locations:
•
•
•
•
Glycolysis – cytosol
Fatty Acid Oxidation: Inner matrix of mitochondria
Citric Acid Cycle: Inner matrix of mitochondria
Oxidative Phosphorylation: Inner membrane of mitochondria
Energy Currency:
Stored in the following
ways:
•
•
•
High energy chemical
species
(i.e. phosphoanhydride
bonds in ATP):
High energy electron
carriers
Membrane potentials
(concentration gradient
and voltage difference)
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Biochemistry I
Lecture 27
Nov 7, 2005
Glycolysis:
• Location: cytosol
• Input: Glucose
• Output: Pyruvate under aerobic, lactate under anaerobic
• Energetics: 2ATP, 2NADH (rapid production of ATP from glucose)
• Key Controlling Step: PFK
• Substrate level phosphorylation
• Direct and Indirect Coupling
Key Enzyme Reactions
A: Hexokinase Reaction (step 1): Glucose + ATP → Glucose-6-P + ADP
Group transfer reaction: Phosphate is transferred from ATP to glucose.
• Traps glucose in the cell as
NH2
G-6-P
H
N
• Favorable hydrolysis of ATP
N
O
H2C
directly coupled to
O
O
O
N
N
O
phosphorylation of glucose.
+
OH
An example of direct
coupling.
O P O P O P O
O
O
O
OH
OH OH
O
O
O
P O
O
CH2
O
OH
ATP
OH OH
OH
OH OH
Energetic coupling: The high
energy of ATP is often used to
drive unfavorable reactions by coupling the favorable hydrolysis of ATP to an unfavorable
second reaction. Consider the following 1/2 reactions:
1st half reaction:
Glucose + Pi → Glucose-6-P
2nd half reaction:
ΔGo = +14 kJ/mol
+ H 2O
ΔGo = - 30 kJ/mol
ATP + H2O → ADP + Pi
______________________________________________________________________
Net sum:
B: Phosphofructokinase (PFK) step 3 [Key regulatory step]:
O
P
O
NH2
O
H
O
CH2
O
H2C
HO
OH
N
N
O
O
+
O
O
O P O P O P O
O
O
O
O
P
N
N
O
O
O
O
P
O
CH2
O
H2C
O
O
O
+
HO
ADP
OH
OH
ATP
OH
OH OH
1st
half
Reaction
F-6-P + Pi + H+ → F-1,6-bisphosphate + H20
2nd
half
Reaction
ATP
H20 → ADP + Pi + H+
ΔG0 = -30 kJ/mol
Net Sum
F-6-P + ATP → F-1,6-bisphosphate + ADP
ΔG0 = -14 kJ/mol
+
2
ΔG0 = +16 kJ/mol
+
ADP
Biochemistry I
Lecture 27
Nov 7, 2005
C. Aldolase (step 4): Fructose-1-6-bisP → dihydroxyacetone
phosphate(DHAP)+glyceraldehyde-3-P (G-3-P).
This is the cleavage of a C6 sugar to give two C3 sugars.
The standard free energy for this reaction is extremely unfavorable ΔGo = +23.8 kJ/mol!
But the Gibbs free energy is actually favorable, ΔG = -6 kJ/mol. Is
this reaction spontaneous?
How is the concentration of G-3-P reduced to insure that the aldolase reaction is
spontaneous?
This completes the “first stage” of glycolysis. Overall Δ G for the first 5 steps under
cellular conditions is -53 kJ/mol. So far, 2 ATP molecules have been consumed.
D. Glyceraldehyde-3-P dehydrogenase (GAPDH) step 6 - Chemical to Redox Energy:
Glyceraldehyde-3-P dehydrogenase: G-3-P + NAD+ → 1,3 bisphosphoglycerate + NADH
This reaction proceeds in two steps. The first step is the oxidation of the aldehyde to the
carboxylic acid (thioester) using NAD+ as the electron acceptor. This results in the
formation of an covalent enzyme intermediate. The second step is the phosphorylation of
the carboxylic acid.
Both reactions are catalyzed by a single enzyme.
G-3-P
HO
O
H
OH
CH2OPO3
H
OPO3
O
OH
CH2OPO3
OH
CH2OPO3
O
H
H O
NH2
+
H2
C
N
O
O
O P
O
OH OH
O
N
O P H
O O 2
C O N
HO
NH2
H2
C
NH2
N
N
N
O
O
O P
O
OH OH
O
N
O P H
2
O O
C O N
OR
HO
NAD+
(Oxidized)
NH2
N
N
OR
NADH
(Reduced)
3
O
1,3 bis
phospho
glycerate
Biochemistry I
Lecture 27
Nov 7, 2005
The Reaction Steps are (see
diagram)
I. ES complex, Cys is deprotonated.
II. Thio group is a nucleophile,
attacks aldehyde, H is transferred to
NAD+ as a hydride ion (H: -), net
transfer of 2 electrons.
III. Stable, thioester intermediate,
NADH is in the reduced form and 3P-G has been oxidized.
IV. Attack of phosphate on stable
acyl-enzyme intermediate,
regenerating enzyme, and producing
1,3-bis phosphoglycerate.
Chemistry
Energetics
1st half Reaction
G " 3 " P # 3 phosphoglycerate + 2e" + H +
(aldehyde) " (carboxylic acid)
"G° = -100 kJ/mol
2nd half
Reaction
NAD+ + 2e" + H + # NADH
"G° = +60 kJ/mol
!
!
NAD + H + G3P " 3PGlycerate + NADH "G° = -40 kJ/mol
!
3PGlycerate + Pi " 1,3bisPGlycerate
"G° = +50 kJ/mol
!
"G° = +10 kJ/mol
!
!
1st Reaction
Sum
+
!
2nd Reaction
!
Overall Sum
+
!
!
E. Phosphoglycerate kinase
O
O
(step 7):
st
Generation of 1 ATP by
substrate level phosphorylation.
(Break-even reaction)
P
O
HO
O
O
P
O
O
O
1,3 bisphosphoglycerate
+
O
O P O P
O
O
ADP
N
N
O
O
OH OH
N
N
O
O
O
O P O P O P
O
O
O
ATP
N
N
O
O
OH OH
+
3-phosphoglycerate
"G° = #19kJ /mol
!
N
N
O
NH2
NH2
!
O
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Biochemistry I
Lecture 27
Nov 7, 2005
F. Pyruvate kinase (step 10):
Generation of Second ATP by a
second substrate level
phosphorylation.
Pi +
phosphoenol
pyruvate
O
O
P
O
H
ADP
ATP
H
H
O
O
H
O
HO
O
O
H
H
H
O
HO
O
enol-pyruvate
H
O
O
pyruvate
"G° = #31.7kJ /mol
!
Regulation of Biochemical Pathways:
General Properties of the Regulation of Biochemical Pathways:
• Step below a convergence point is usually regulated.
• Usually steps that involve a large negative ΔG are regulated, as these are usually
irreversible.
• Opposing pathways are coordinately regulated, usually at a single key step.
Mechanisms of Regulation:
• Change in levels of enzymes by regulation of the synthesis/degradation. (slow)
• Change in the activity of enzymes by covalent modification (e.g. phosphorylation) of the
enzyme (moderately fast)
• Change in the activity of enzymes by feedback inhibition by a chemical that is near the
end of the pathway, or in another pathway (Usually allosteric activators/inhibitors). This is
fast.
• Product inhibition. This is very fast.
Regulation of Glycolysis:
Glucose ⇔ G-6-P
Hexokinase:
Inhibited by G-6-P
Fructose-6-P ⇔ F-1,6-P
Phosphofructose kinase (PFK)
Activated:
ADP, AMP
F2,6-P
cAMP
PEP⇔Pyr
Inhibited:
ATP
Citrate (TCA)
PEP (Glycolysis)
Pyruvate Kinase:
Activated:
Inhibited:
AMP,
ATP,
F-1,6-P
acetyl CoA, alanine
5
Pi
enol-pyruvate
H
O
+
Biochemistry I
Lecture 27
6
Nov 7, 2005