Download 27. GE_7.27 Gluconeo.. - College of Pharmacy at Howard University

BIOCHEMISTRY COURSE –
PHARMACY BIOMEDICAL PREVIEW
PROGRAM, SUMMER 2016
Ginika Ezeude, B.A. Biochemistry
Barnard College of Columbia University
2nd Year Pharmacy Student
Contact: [email protected]
METABOLIC PATHWAYS
1. Glycolysis
2. Gluconeogenesis
3. Pentose Phosphate Pathway
In animals and vascular plants, glucose has three major fates:
1. It may be stored (as a polysaccharide or as sucrose);
2. Oxidized to a three-carbon compound (pyruvate) via glycolysis to
provide ATP and metabolic intermediates;
3. Or oxidized via the pentose phosphate (phosphogluconate)
pathway to yield ribose 5-phosphate for nucleic acid synthesis
and NADPH for reductive biosynthetic processes
1. GLUCONEOGENESIS
- Comparison between Glycolysis & Gluconeogenesis
- Regulation of Gluconeogenesis
GLUCONEOGENESIS
The important precursors of glucose in
animals are three-carbon compounds such as
lactate, pyruvate, and glycerol, as well as
certain amino acids.
Organisms need a method for synthesizing
glucose from non-carbohydrate precursors;
this is accomplished by gluconeogenesis.
Cori Cycle
CHECK POINT QUESTION
What enzyme(s) are used to convert pyruvate to
phosphoenolpyruvate in gluconeogenesis?
A.) Pyruvate kinase
B.) Pyruvate kinase and pyruvate carboxylase
C.) Pyruvate Carboxylase and PEP carboxykinase
D.) PEP carboxykinase and pyruvate kinase
GLUCONEOGENESIS
Step 10. The first of the bypass reactions
in gluconeogenesis is the conversion of
pyruvate to phosphoenolpyruvate
(PEP). Pyruvate carboxylase, a
mitochondrial enzyme, converts the
pyruvate to oxaloacetate. Mitochondrial
malate dehydrogenase reduces
oxaloacetate formed from pyruvate to
malate. Malate is then re-oxidized to
oxalate. Then mitrochondrial PEP
carboxykinase converts Oxalate to PEP.
GLUCONEOGENESIS
Step. 3 The second glycolytic reaction that cannot participate in gluconeogenesis is the
phosphorylation of fructose 6-phosphate by PFK-1. The reaction is catalyzed by a different
enzyme, Mg2-dependent fructose 1,6-bisphosphatase (FBPase-1), which promotes the
essentially irreversible hydrolysis of the C-1 phosphate.
CHECK POINT QUESTION
Mitochondrial malate dehydrogenase utilizes which of the following
cofactors when converting oxaloacetate to malate?
A.) NAD+
B.) NADH
C.) FADH2
D.) CO2
GLUCONEOGENESIS
Step. 1. The third bypass is the final reaction of gluconeogenesis, the dephosphorylation of
glucose 6-phosphate to yield glucose. Reversal of the hexokinase reaction would require
phosphoryl group transfer from glucose 6-phosphate to ADP, forming ATP, an energetically
unfavorable reaction. The reaction catalyzed by glucose 6-phosphatase does not require
synthesis of ATP; it is a simple hydrolysis of a phosphate ester.
REGULATION OF
GLUCONEOGENESIS
Pyruvate can be converted either to
acetyl-CoA (by the pyruvate
dehydrogenase complex; to fuel the
citric acid cycle, or to oxaloacetate (by
pyruvate carboxylase) to start the
process of gluconeogenesis.
1. Acetyl-CoA is a positive allosteric
modulator of pyruvate
carboxylase and a negative
modulator of pyruvate
dehydrogenase.
REGULATION OF
GLUCONEOGENESIS
2. The second control
point in gluconeogenesis
is the reaction catalyzed
by FBPase-1 which is
strongly inhibited by AMP
CHECK POINT QUESTION
Acetyl CoA stimulates which enzyme in the regulation of
gluconeogenesis?
A.) pyruvate carboxylase
B.) pyruvate kinase
C.) pyruvate dehydrogenase complex
D.) All of the Above
REGULATION OF GLUCONEOGENESIS
Fructose 2,6-Bisphosphate Is a Potent Regulator of Glycolysis and
Gluconeogenesis
The hormonal regulation of glycolysis and gluconeogenesis is mediated by fructose 2,6bisphosphate, an allosteric effector for the enzymes PFK-1 and FBPase-1. Fructose 2,6bisphosphate activates PFK-1 and stimulates glycolysis in liver and, at the same time, inhibits
FBPase-1, thereby slowing gluconeogenesis.
REGULATION OF FRUCTOSE 2,6-BISPHOSPHATE LEVEL
Fructose 2,6-bisphophate is formed by phosphorylation of fructose 6-phosphate,
catalyzed by phosphofructokinase- 2 (PFK-2), and is broken down by fructose 2,6bisphosphatase (FBPase-2).
CHECK POINT QUESTION
Glucagon and Insulin regulate the balance of which enzymes?
A.) FBPase-1 and PFK-1
B.) FBPase-2 and PFK-2
C.) PFK-1 and PFK-2
D.) FBPase-1 and FBPase-2
WHEN IS GLUCONEOGENESIS HAPPENING?
● Gluconeogenesis creates glucose from non-carbohydrate
precursors
● GNG occurs when there are no sources of sugar
o No sugar in the blood
o All glycogen (glucose polymer, stored in liver/muscles) has
been used up
o Body can use waste materials from other cycles/other food
metabolism (lipids mostly)
● In fasting or starvation states, all generated glucose is first
transported to the brain
2. TCA CYCLE
- Products obtained from citric acid cycle
PYRUVATE -- NOT JUST LEFTOVERS
● Pyruvate is an important molecule in its own right
● From glycolysis, it is “charged” with CoA (resulting in 1 molecule CO2, 1
NADH and 1 Acetyl-CoA) and moves onto the Citric Acid Cycle, or the
Kreb’s Cycle
● This cycle takes AcetylCoA and generates 1
GTP, 3 NADH, 1
FADH2 per Pyruvate
● How many Pyruvates
result from Glycolysis?
CITRIC ACID CYCLE
● Acetyl-CoA (2C) joined with Oxaloacetate (4C) to make
Citrate (6C)
o
“Citric acid cycle” name is deserved!
● Carbons are swapped around and bonds are oxidized and
reduced, until…
● The two extra C’s are chopped off as 2 CO2
● Another Acetyl-CoA is added, cycle resumes
o
o
Each molecule of glucose produces how many pyruvates?
How many times must the TCA turn to process one glucose
molecule?
CITRIC ACID CYCLE COMPONENTS ARE IMPORTANT
BIOSYNTHETIC INTERMEDIATES
THINGS TO LEARN FROM TCA
● TCA has 8 steps and plenty of intermediates
o these ~8-6 carbon molecules are used for many structures and
functions in the body
 Ex: alpha-ketoglutarate is important for the synthesis and
maintenance of amino acid pathway; it is a nitrogen shuttle and
supplies aminos for biosynth of AA
o However, while it is convenient to snatch pre-made molecules from an
ongoing cycle, the cycle still needs to keep enough intermediates to
function!
 Anaplerosis is the process of replenishing TCA cycle
intermediates that have been extracted for biosynthesis
 What does this word look like? (HINT: ANA)
o Biochemical pathways MUST BE BALANCED or else someone is
going to run out of energy and/or necessary molecules
TCA = END OF THE ROAD
● Glucose goes through glycolysis then the end-products,
2 pyruvate, go through the TCA cycle
● What do we get out of this?
ENERGETIC YIELD OF GLUCOSE MONOMER
● Glycolysis
o 2 ATP
o 2 NADH
 2*3 = 6 ATP
o 2 Pyruvate
 Citric Acid Cycle
● 2 NADH from 2
transition rxns
o
6 ATP
● 6 NADH (2 turns)
o
18 ATP
● 2 FADH2 (2 turns)
o
4 ATP
● 2 GTP (2 turns)
Final Count:
● 8 ATP from glycolysis
● 30 ATP from 2
pyruvates in the citric
acid cycle
● 38 ATP from 1
molecule of glucose
CITRIC ACID: INS AND OUTS
● What goes in:
o
o
o
o
o
Acetyl-CoA
H2O
3NAD+
1 FAD
1 GDP
● What comes out:
○
○
○
○
2 CO2
3 NADH
1 FADH2
1 GTP
METABOLIC COFACTORS: NOTE
● You’ve seen NAD+/NADH floating around, right?
o They carry H’s around to reduce and oxidize molecules for
energy
o Also carry those electrons/H’s to the OxPhos Pathway, which
we’ll talk about next class
o FADH2 functions similarly, can “extract” 2 H
o However, NAD+ has a cousin…
● NADH is a catabolic (energy-generating) cofactor
o Whenever you see NADH in a pathway, you know the body is
breaking down molecules for energy
● NADPH is an anabolic (biosynthetic) cofactor
o Whenever you see NADPH in a pathway, you know that the
body is conducting biosynthesis and some molecule is being
made
o A good example of an anabolic pathway is the PPP...
3. PENTOSE PHOSPHATE
PATHWAY
- Oxidative Phase
- Non-Oxidative Phase
PENTOSE PHOSPHATE PATHWAY
Pentose
Phosphate
Pathway involves
the oxidation of
glucose 6phosphate to
pentose
phosphates. In
this oxidative
pathway, NADP is
the electron
acceptor, yielding
NADPH.
OXIDATIVE PHASE OF PENTOSE PHOSPHATES PATHWAY
Two molecules of
NADPH is generated
NON-OXIDATIVE PHASE OF PENTOSE
PHOSPHATES PATHWAY
In this non-oxidative phase, ribulose 5phosphate is first epimerized to xylulose 5phosphate
NON-OXIDATIVE PHASE OF PENTOSE
PHOSPHATES PATHWAY
CHECK POINT QUESTION
How many NADPH’s are generated from the oxidative phase of the
pentose phosphate pathway?
A.) 1
B.) 2
C.) 4
D.) None, only NADH is generated
PRINCIPLES OF METABOLIC REGULATION
How do cells to maintain a balance even in the face of outside perturbation?
COORDINATED REGULATION OF GLYCOLYSIS
AND GLUCONEOGENESIS
Hexokinase Isozymes of Muscle and Liver Are Affected Differently by Their Product, Glucose 6Phosphate
 Hexokinase, which catalyzes the entry of free glucose into the glycolytic pathway, is a
regulatory enzyme.
 There are four isozymes (designated I to IV).
 Hexokinase II has a high affinity for glucose
 Muscle hexokinases I and II are allosteric ally inhibited by their product, glucose 6phosphate, so whenever the cellular concentration of glucose 6-phosphate rises above its
normal level, these isozymes are temporarily and reversibly inhibited, bringing the rate of
glucose 6-phosphate formation into balance with the rate of its utilization and re-establishing
the steady state.
What mechanism does this sound like?
COORDINATED REGULATION OF
GLYCOLYSIS AND GLUCONEOGENESIS
Comparison of the kinetic properties of hexokinase IV (glucokinase) and
hexokinase I.
PHOSPHOFRUCTOKINASE-1 IS UNDER
COMPLEX ALLOSTERIC REGULATION
 The metabolically irreversible reaction catalyzed by PFK-1 is the step that commits
glucose to glycolysis.
 In addition to its substrate-binding sites, this complex enzyme has several
regulatory sites at which allosteric activators or inhibitors bind.
 ATP is not only a substrate for PFK-1
but also an end product of the glycolytic
pathway. When high cellular [ATP]
signals that ATP is being produced
faster than it is being consumed, ATP
inhibits PFK-1 by binding to an
allosteric site and lowering the affinity of
the enzyme for fructose 6-phosphate.
PYRUVATE KINASE IS ALLOSTERICALLY
INHIBITED BY ATP
High concentrations of ATP, acetyl-CoA, and long-chain fatty acids,
allosterically inhibit all isozymes of pyruvate kinase and the accumulation
of fructose 1,6-bisphosphate triggers its activation.
CHECK POINT QUESTION
Which of the following is NOT an allosteric inhibitor of Pyruvate
Kinase?
A.) Long chain fatty acids
B.) ATP
C.) NADH
D.) Acetyl CoA
THANK YOU!
Next: Enzyme Kinetics