Pyruvate dehydrogenase and TCA cycle -Mitsouras

Question 1

Products of the TCA cycle, 1 full round

Answer 1

2 CO2
1 GTP
3 NADH
1 FADH2
No net production or consumption of intermediates
intermediates are replenished via anaplerotic reactions

every glucose goes through TWO turns of the TCA cycle

Question 2

Coenzymes of TCA cycle

Answer 2

Function as the electron carriers in their reduced forms (NADH, NADPH, FADH2) to the Electron transport chain

enzymes with “dehydrogenase” in their names mean that NADH or FADH2 are produced

NADH and NADPH:
2 electron donor/acceptor
NADH–> catabolic pathways
NADPH–> reductive biosynthesis
both are diffusible and NOT permanently attached to enzymes.
*NAD+ and NADP+ are biologically active forms of niacin/nicotinic acid (vitamin B3)

FAD/FADH2:
2 electron donor/acceptor
involved in catabolic pathways
has a prosthetic group that is permanently attached to enzymes.
biologically active forms of riboflavin (vitamin B2)

Question 3

Niacin Deficiency

Answer 3

NAD+ and NADP+ are the biologically active forms of niacin/nicotinic acid (vitamin B3)

Deficiency causes pellagra

symptoms: 3 D’s
- Dermatitis (photosensitive) (required for the repair of skin)
- Diarrhea (GI)
- Dementia (depression, apathy, headache, fatigue, loss of memory) (CNS)

was common when corn was a dietary staple now only seen in chronic alcoholics and in individuals with disorders of tryptophan metabolism (niacin can be synthesized from tryptophan)

Question 4

Riboflavin Deficiency

Answer 4

FAD & FMN are biologically active forms of riboflavin (vitamin B2)
Dietary sources => milk, eggs, liver (animal products–> consider pt diet if appear w these symptoms)
Riboflavin deficiency causes ariboflavinosis
Symptoms:
- Dermatitis
- Cheiliosis (cracking in corners of lips)
- Glossitis (magenta tongue /swelling)
usually accompanied by other nutrient deficiencies
rare in industrialized countries, sometimes seen in chronic alcoholics

Question 5

Pyruvate –> acetyl CoA

Answer 5

Pyruvate (cytoplasm) –> pyruvate (mitochondrial matrix) via pyruvate transporter

Pyruvate –> AcCoA by Pyruvate Dehydrogenase Complex (PDH)

• oxidative phosphorylation
• irreversible and highly regulated

Question 6

pyruvate dehydrogenase structure and cofactors

Answer 6

PDH is a multi-subunit enzyme: 3 catalytic subunits (E1-3)–> require 5 cofactors for activity, and 2 regulatory subunits (PDH kinase and phosphatase)

Feedback inhibition:
acetyl CoA and NADH inhibit the conversion of pyruvate–> Acetyl CoA

Cofactor requirements for PDH:
E1==> TPP (thiamine pyrophosphate)
E2==> lipoid acid (target of arsenic poisoning)
E3==> FAD
NAD+ and CoA are other required cofactors

TPP, lipoid acid and FAD are prosthetic groups (permanently attached to enzyme)
NAD+ and CoA are diffusible cofactors

Question 7

PDH deficiency

Answer 7

Disease from a mutation in usually E1
will prevent pyruvate from being converted to Acetyl CoA and will allow it to enter the lactic acid cycle instead (LDH activity depends on the pyruvate/lactate ratio)–> build up of lactic acid causes lactic acidosis and neurological symptoms

X-linked inheritance
Broad clinical spectrum of symptoms & severity proportional to residual activity of mutant enzyme but all share neurological & metabolic symptoms
 Most severe:
	- Mental retardation
	- Spasticity
	- Seizures
	- Structural abnormalities in CNS

Treatment: management of symptoms via nutritional & pharmacological intervention

- ketogenic diet (low carbohydrate/high fat) glucose can’t be used by brain--> use ketone bodies. ketogenic diet=get energy from ketones from fats (ATP)
- thiamine administration (increase the activity of the enzyme) 
- muscle relaxants & anti-convulsants (manage symptoms)

Question 8

Thiamine Deficiency

Answer 8

Vitamin B1 causes:
1. Beriberi:
caused by thiamine deficiency in areas where polished rice is the major component of the diet
-can be wet or dry:
- wet: edema & cardiovascular symptoms (cardiac failure)
- dry: muscle wasting & peripheral neurologic dysfunction

2. wernicke-korsakoff syndrome:
   In industrialized countries thiamine deficiency observed in chronic alcoholics & gives rise to neuropsychiatric syndromes (EtOH interferes with thiamine absorption)
   - Wernicke encephalopathy: reversible (ataxia, confusion, nystagmus) if progresses can cause Korsakoff
   - Korsakoff syndrome: irreversible (deficits in learning & memory)
   - Wernicke-Korsakoff syndrome: chronic and prolonged (years) deficiency –> causes psych problems

thiamine also required for transketolase (HMP) and alpha ketoglutarate dehydrogenase (TCA cycle)

Question 9

Regulation of PDH

Answer 9

1) allosteric effectors on PDH catalytic subunits
- inhibition by Acetyl CoA and NADH
2) regulation of catalytic subunits by PDH kinase & PDH phosphatase (regulatory subunits of PDH)

-high energy charge and product accumulation (NADH, acetyl CoA and ATP) –>PDH kinase on==> phosphorylates PDH==> INACTIVATES PDH

-low energy charge and substrate accumulation (ADP, pyruvate, Ca2+)–> PDH kinase=OFF
PDH phosphatase ON==> dephosphorylates PDH ==> ACTIVATES PDH

-insulin stimulates PDH phosphatase ==> activates PDH

• NADH and Acetyl CoA are 2 fold in action:
• directly inhibit via negative feedback of PDH kinase and by allosteric regulation of PDH kinase

Question 10

CoA and Pantothenic Acid Deficiency

Answer 10

Synthesized from pantothenate/pantothenic acid
Dietary sources=> most foods but eggs & yeast are good sources
Deficiency very very rare;
Causes burning foot syndrome (observed in POWs in Asia during WWII)
Symptoms:
- painful burning feet
- irritability, restlesness
- fatigue, apathy, sleep disturbances
- GI distress, nausea, vomiting, abdominal cramps

CoA synthesized from vitamin B5

Question 11

TCA Reactions requiring cofactors

Answer 11

alpha ketoglutarate –> succinyl-CoA + CO2 by alpha ketoglutarate dehydrogenase
-requires Thiamine, lipoic acid, FAD, NAD+ & CoA

Question 12

Regulation of the TCA cycle

Answer 12

• citrate synthase: inhibited by citrate and ATP
• Isocitrate dehydrogenase: inhibited by NADH & stimulated by ADP & NAD+
• a-ketoglutarate dehydrogenase: inhibited by succinyl-CoA, NADH & ATP
  –> Net effect => Low energy charge ON
  High energy charge OFF
  OFF when intermediates accumulate

Question 13

Anaplerotic reactions of TCA cycle

Answer 13

anaplerotic reactions regenerate intermediates
2 types:
1. amino acid degradation replenished 4C and 5 C acids
(fumarate–> AAs, oxaloacetate–> aspartate, alphaketoglutamate–> glutamate, succinyl-CoA–> AAs)

2. oxaloacetate regenerated from pyruvate from pyruvate carboxylase –> needed for gluconeogensis
   - occurs in the liver and kidney
   - requires biotin as a cofactor

Question 14

Biotin deficiency

Answer 14

Pyruvate carboxylase requires biotin as a cofactor
Biotin deficiency caused by eating raw eggs (avidin)
Deficiency symptoms:
- Dermatitis
- Glossitis
- Loss of appetite