Lecture 19 Flashcards
where does cellular respiration occur?
- Glycolysis – cytosol
* Respiration – mitochondria
where does ATP generation occur?
inner membrane of mitochondrion
where does pyruvate oxidation and CAC occur?
matrix of mitochondrion
cristae
folds in the inner mitochondrial membrane that project into the mitochondrial matrix. The enzymes of the ETC and oxidative phosphorylation are located mainly on the cristae
how is pyruvate transported from the cytosol into the mitochondria? and what is it converted to?
- mitochondrial pyruvate carrier (MPC)
- converted to acetyl CoA
- pyruvate is a “mono carboxylate”
rxn for conversion to acetyl CoA
pyruvate + CoA + Nad+ —> Acetyl CoA + CO2 + NADH
what is an oxidative decarboxylation catalyzed by?
pyruvate dehydrogenase complex
what family does pyruvate dehydrogenase complex belong to?
family of α-keto Dehydrogenases
what is the ΔGº’ of the pyr–> CoA rxn?
Pyruvate + CoA + NAD+ —> Acetyl CoA + CO2 + NADH
-33.5 kJ/mol
What three enzymes catalyze the 5 rxns
E1- pyruvate dehydrogenase
E2- dihydrolipoamide transacetylase
E3- Dihydrolipoamide dehyrdogenase
The 3 enzymes require which 5 cofacors?
- Thiamine Pyrophosphate (TPP)
- Lipoic Acid
- Coenzyme A (CoA or CoA-SH)
- Flavin Adenine Dinucleotide (FAD)
- Nicotinamide Adenine Dinucleotide (NAD+)
which cofactor stabilizes carboanions?
thiamine pyrophosphate
thiamine (vitamin b1)
The 3 enzymes require which 5 cofactors?
- Thiamine Pyrophosphate (TPP)
- Lipoic Acid
- Coenzyme A (CoA or CoA-SH)
- Flavin Adenine Dinucleotide (FAD)
- Nicotinamide Adenine Dinucleotide (NAD+)
which cofactor stabilizes carboanions?
thiamine pyrophosphate
thiamine (vitamin b1
what are coenzymes?
Coenzymes are small molecules. They cannot by themselves catalyze a reaction but they can help enzymes to do so. In technical terms, coenzymes are organic nonprotein molecules that bind with the protein molecule (apoenzyme) to form the active enzyme (holoenzyme)
Pyruvate Dehydrogenase (PDH) what are the methods to identify
is a large multisubunit complex.
Use Crystallography and NMR to solve structures of domains, subunits, and enzymes
Use Electron Microscopy to determine how they are organized
Not easy – Complex is somewhat flexible
Pyruvate Dehydrogenase Complex
What’s the advantage?
- Substrate Channeling- int. of multistep pathway are handed off form one active site to the next w/o diffusing complex- makes smooth
- Effective Concentration-
- A + B <==> A-B + A-B
- tethering can increase the effective concentration
• Regulation-
TPP- Location and function
- tightly bound to E1
- decarboxylates pyruvate, yielding hydroxyethyl- TPP
Lipoic Acid- location and function
- covalently bound to E2 via lysine (“swinging arm”)
- accepts hydroxyl carbanion from TPP as acetyl group
Coenzyme A (CoA)- location and function
- dissociable substrate for E2
- accepts acetyl group from lipoamide
FAD- location and function
- tightly bound to E3
- accepts pair of e- from reduced lipoamide
NAD+ - location and function
- dissociable substrate for E3
- accepts pair of e- from reduced FADH2
Enzymes of the Krebs cycle
Pyruvate Dehydrogenase & α-Ketoglutarate Dehydrogenase are in the same enzyme family.
ReWhat is PDH inhibited by?
acetyl CoA, NADH
compete for binding with substrates CoA and NAD+
what is PDHG regulated by?
covalent modifcation:
• PDH kinase (inhibits) (phosphorylates E1)
–kinase activated by NADH and acetyl CoA
–kinase inhibited by NAD+ and pyruvate
• PDH phosphatase (activates)
–phosphatase activated by insulin and Ca2+
Transcriptional regulation of PDH kinase
Expression of many glucose metabolism genes is regulated by a specific transcription factor HIF-1.
low O2–> stabilized HIF –> DNA –> RNA–> Protein –> PDH Kinase
what is HIF-1 and what is it stabilized by?
transcriptional factor
hypoxia
hydroxylation
is a signal to degrade HIF-1 by a proteolysis pathway (ubiquitin pathway)
HYPOXIA (low O2) –> HIF1 stabilized
low or high
PDH PDH activity pyruvate consumed acetyl CoA made glycolytic enzymes CAC activity
More PDH Kinase PDH Phosphorylation Increases PDH Activity Decreases Less Pyruvate is consumed Less Acetyl CoA is made More glycolytic enzymes made Less Citric Acid Cycle Activity
More lactic acid production rxn:
Pyruvate + NADH —-> Lactate + NAD+
Hypoxia-Inducible Factor (HIF) is important for:
-(HIF-1 also stimulates production of specific types of Growth Factors (VEGF and EPO))
•Adaptation to high altitudes
•Survival and growth of cells in low O2 environments.
•Tumor growth
VEGF
(Vascular Endothelial Growth Factor and Erythropoietin)
VEGF stimulates formation of new blood vessels.
• EPO stimulates production of new erythrocytes
Erythropoietin (“EPO”)
- made in kidneys (and liver)
- heavily glycosylated
- stimulates survival of red blood cell progenitors
The “JAK/STAT” pathway is another type of signaling pathway that uses reversible phosphorylation of proteins.
high altitude training
“Through the production of normobaric hypoxic (oxygen reduced) air, we can simulate altitudes of up to 21,000ft/6,400m. As a result, athletes, fitness enthusiasts and health conscious individuals worldwide can take advantage of the benefits associated with altitude training while at sea-level”.
too much EPO can be dangerous - WHY?
increase the thickness (viscosity) of the blood, raising the risk for blood clots, heart attacks, and strokes
key concepts for PDH- Regulation
• Regulation
– Inhibition by products (NADH and Acetyl CoA)
– Phosphorylation of Pyruvate Dehydrogenase
– Regulation of PDH Kinase
– Regulation of expression
– Mechanism by which HIF senses Oxygen
When glucose is converted to ethanol by yeast (as in the production of beer) how much of the chemical energy that was originally stored in glucose still remains in the end product, ethanol, that is produced?
92%
Which one of the following molecules has the highest molecular weight?
Acetyl CoA
When Acetyl CoA reacts with oxaloacetate in the Citrate Synthase reaction how many carbons are in the product, citrate that is formed?
5
Which ONE of the following is NOT a coenzyme or substrate involved in the pyruvate dehydrogenase complex reaction?
ATP`
Which ONE of the following provides a long arm that can swing a substrate from one site to another in the PDH complex?
Lipoamide
To get you to look carefully at these structures – answer this - which ONE has the highest molecular weight?
FAD
Which part of the PDH complex is affected most directly by Arsenite?
FAD
