bioc 311

Thiamine (for TPP), Panthotenic acid (Coenzyme A), Riboflavin (FAD), and Niacin (NAD+)

Lipoyllysyl arm: can act as a arm that swings the reduced product between E1 and E3.

- Active sites are close to one another: distance traveled to next active site is reduced, enhanced enzymatic rate.
- Intermediates are channeled to the other enzymes, minimizes side reactions
- Regulation mechanisms simultaneously regulate activities of the entire complex

E1: Pyruvate dehydrogenase
E2: Dihydropipoyl transacetylase
E3: Dihydrolipoyl dehydrogenase

Berberi: loss of neuronal functions; the brain needs energy from oxidation of glucose, therefore effective PDC is important in the brain

1) Decarboxylation
2) Oxidation of the keto group on C2 to carboxyl
3) Activation by linkage to CoA through thioester bond

CO2 diffuses out of mitochondria

Dihydrolipoyl dehydrogenase (in its oxidized form)

Pyruvate + CoA + NAD+ ------> Acetyl CoA + CO2 + NADH

NAD+

PDC and Alpha-ketoglutarate dehydrogenase

Binds covalently to sulhydryl compounds: forms bidentate adduct with disulfide group in lipoamide, blocks PDC and Alpha-KDH

Structural analog of Phosphate (PO4^3- vs AsO4^3-), competes with it at 5th step in glycolysis, giving 1-arseno-3-phosphoglycerate instead of 1,3-biphosphoglycerate. Therefore, you get 3PG WITHOUT the normally produced ATP.

Pyruvate dehydrogenase complex (PDC) and Lactate Dehydrogrnase (LDH); They lead to Aerobic oxidation or Anaerobic homolactic fermentation.

PDC's activity is suppressed by phosphorylation: pyruvate is converted into lactate instead. Ind FDG-PET scan, we can see a lot of radioactively labeled glucose taken up, as ATP is generated only from anaerobic homolactic fermentation.

high concentrations of NADH and Acetyl CoA compete with NAD+ and CoA on their respective enzymes' binding site: they reverse reactions 3 and 5, and prevent needless conversion of pyruvate into Acetyl CoA.

Activators: Acetyl-CoA, NADH
Inhibitors: Pyruvate, ADP, Ca2+, high Mg2+, K+

Phosphorylates E1, making it inactive. Shuts down PDC

Mg2+ and Ca2+.
It removes the phosphate on inactive E1

Citric Acid Cycle
Krebs Cycle
Tricarboxylic Acid Cycle

...there is both catabolism and anabolism in the CAC

Cataplerotic

Anaplerotic

1) Produce reducing equivalents (NADH, FADH2), whose oxidation lead to ATP synthesis through oxphos. 1 ATP equivalent is also made in one turn (GTP).
2) Produce organic intermediates used in biosynthesis
3) Eliminate surplus organic compounds as CO2

NO

The Hydrogen gradient between the intermembrane space and the mitochondrial matrix.

Oxaloacetate + Acetyl-CoA + H2O ------> CoASH + Citrate

2 CO2, 3 NADH, 1 FADH2 and 1 GTP

Energy gets trapped in compounds like NADH and FADH2, but they get used in ETC to produce energy

Energy trapped in GTP or ATP: energy is trapped in high energy bond of a molecule. Used as energy source for enzymatic reactions later on.

1: Citrate synthase
3: Isocitrate
4: alpha-ketoglutarate dehydrogenase

Hydrolysis of Thioester bond in S-Citryl-CoA: ∆G°' = -31.5kJ/mol

pro S-arm

pro R-arm

Isomerization. The reaction is reversible

intermediate : cis-Aconitate
final product : Isocitrate

Fluoroacetate can get converted into Fluorocitrate, which is toxic. Inhibits aconitase and blocks citrate from going in and out of mitochondria

It regulates PFK

...Alpha-Ketoglutarate

Isocitrate dehydrogenase

1 NADH and 1 CO2

Amino Acids

Succinyl-CoA

1 NADH and 1 CO2

Pyruvate dehydrogenase complex

E1: alpha-ketoglutarate dehydrogenase
E2: dihydrolipoyl transsuccinylase
E3: dihydrolipoyl dehydrogenase

Succinate and GTP are made from Succinyl-CoA; Sunninyl-CoA synthetase is used in this reaction

The energy is converted to a high energy P-P bond in GTP: this process of energy conversion is called Covalent energy transduction.

Pi + Succinyl-CoA + GDP ---> Succinate + GTP + CoASH

GTP is converted into ATP with ADP and Nucleoside diphosphate kinase.

Succinate ---> Fumarate

Succinate dehydrgenase is used for the 6th reaction. FADH2 is made besides Fumarate.

It is the only enzyme not located in the matrix, but instead located in the Inner Mitochondrial Membrane (IMM).

Because it is covalently linked to HIS residue in succinate dehydrogenase.

Malonate is a homolog of succinate (malonate : -OOC-CH2-COO-): competitive inhibitor of SDH; inhibits FORWARD and REVERSE reactions

It's cyclical nature: was added to muscle homogenates w/ different substrates in the CAC, always ended up with accumulation of Succinate.

Hydration of the C=C double bond to generate Malate; fumarase is the enzyme used.

Fumarate is converted to Oxaloacetate; malate dehydrogenase is used.

The reaction after Oxaloacetate production is highly exergonic; therefore, the coupling of these 2 reactions makes the 8th reaction possible. 3rd and 4th reaction keep CAC running unidirectionally.
Also, since 1st reaction i physiologically favourable, there is very little oxaloacetate; therefore reverse reaction of 8 occurs rarely.

Even though Citrate does not appear to contain an asymmetric carbon, it behaves as if it contained a chiral centre.

Yes they are distinguishable: if you radiolabel the carboxymethyl group of oxaloacetate, after one turn of the cycle, there will always be the same CO2 radiolabeled produced.

RE FACE

SI FACE

The SI face

It will be transformed into CO2 only after 2 cycles, either in conversion of Isocitrate to alpha-ketoglutarate, or conversion of the later into Succinyl-CoA.

Only after the 3rd turn will CO2 possibly be made from the methyl carbon brought by Acetyl-CoA: half of the time it will be converted in the 3rd turn, then 1/4 of the time in the 4th turn, and so on.

high ratios of NADH/NAD+, ATP/ADP, Acetyl-CoA/CoASH : if they are high, it means there is enough energy in the cell, CAC is therefore downregulated

Low energy charge...

PDC (which makes pyruvate) and Pyruvate Carboxylase (which makes Oxaloacetate)

1) Oxidation for ATP synthesis
2) Conversion to starting material for biosynthesis (Oxaloacetate)

Since we can use metabolites found in CAC for biosynthesis of biological molecules, we need to create Oxaloacetate to replace the "deviated" product of CAC and continue the cycle

1) Citrate Synthase
2) Isocitrate dehydrogenase
3) Alpha-Ketoglutarate dehydrogenase

1) Substrate availability
2) Product inhibition
3) Competitive feedback inhibition
4) Allosteric activation
5) Allosteric inhibition

NADH

Substrate availability: Acetyl-CoA and Oxaloacetate.
Product inhibition: Citrate.
Competitive feedback inhibition: Succinyl-CoA.
Allosteric inhibition: NADH.

Product inhibition: NADH
Allosteric inhibition: ATP
Allosteric activation: ADP and Ca2+

...Citrate

Inhibits PFK, activates Acetyl-CoA carboxylase, increase FA synthesis

ATP + Citrate + CoA ---(ATP Citrate lyase)---> ADP + Pi + Oxaloacetate + Acetyl-CoA

Product inhibition: inhibited by NADH, Succinyl-CoA.
Allosteric activation: Ca2+

1) Positive effects on biosynthesis
2) Downregulation of glycolysis

ATP

1) Anaerobic lactic fermentation
2) Aerobic oxidation
3) Anaerobic alcoholic fermentation

NAD+ and lactate

NAD+, CO2 and water

NAD+, CO2m and EtOH

1) Add phosphoryl groups to glucose
2) Chemically convert phosphorylated intermediates into compounds with high phosphoate group-transfer potentials
3) Chemically couple the subsequent hydrolysis of reactive substances to ATP synthesis

2 ATP

~38 ATP

Na+ - glucose symport; no energy is required for this transporter.

Through a glucose uniport: this is a passive transport.

1) Add phosphoryl groups to glucose
2) Chemically convert phosphorylated intermediates into compounds with high phosphoate group-transfer potentials
3) Chemically couple the subsequent hydrolysis of reactive substances to ATP synthesis

2 ATP

~38 ATP

Na+ - glucose symport; no energy is required for this transporter.

Through a glucose uniport: this is a passive transport.

SGUT-1

Liver, pancreatic ß cells, small intestine, kidney.
Transports glucose, galactose and fructore. Low affinity, high capacity; serves as glucose sensor in ß cells of pancreas.

GLUT-4: skeletal and cardiac muscles, adipocytes

Glucose + ATP ---(Mg2+)---> Glucose-6-phosphate + ADP + H+

Hexokinase

Enzyme that transfers phorphoryl groups between ATP and a metabolite.

The divalent metal shields negative charges of oxygen in phosphate groups, making phosphorus more accessible for nucleophilic attack of C6-OH group of glucose.

Glucose-6-phosphate

Glucokinase

maintenance of blood glucose level

Hexokinase I (Km: 0.03mM vs 5mM)

LECTURE 3

1. adenylate kinase regeneration of ATP, leading to a fourfold increase of AMP, increasing PFK activity.
2. Substrate cycling due to PFK-1 and FBPase-1

Activator: PFK-1
Inhibitor: FBPase-1

alpha pancreatic cells

Increase production of cAMP

cAMP activates PKA, which will phosphorylate PFK-2 and FBPase-2.

PFK-2: phosphorylation INHIBITS conversion of F1P into F-2,6-P.
FBPase-2: phosphorylation INCREASES dephosphorylation activity on F-2,6-P

activity of PFK-1 will be reduced as F-2,6-P concentration are decreasing

DECREASED glycolytic activity due to PFK-1 activity inhibition.

LECTURE 4

A greater intake in refined carbohydrates explains this

-80% fiber content
-30% protein content
+10% calorie density

High Fructose Corn Syrup

Positive

glucose units

Galactose + Glucose

Glucose + Fructose

1. Used for energy production (incorporated into glycolysis)
2. Converted to glycogen
3. Used for synthesis of fat

In the liver

Fructose-6-phosphate: used at the 3rd step

Glyceraldehyde-3-phosphate: used at the 6th step

Hexokinase

in muscle: it can clease fructose-1,6-biphosphate

in the liver: it can cleave Fructose-1,6-biphosphate AND fructose-1-phosphate

glyceraldehyde and dihydroxyacetone phosphate

an excess of lipids

muscle

genetic disorder due to a lack of Fructose-1-phosphate aldolase

buildup of fructose-1-phosphate: there is depleting of ATP and Pi

a fructose free diet

Galactokinase

Galactose-1-phosphate uridylyl transferase

UDP-galactose-4-epimerase

Phosphoglucomutase

glucose-1-phosphate, UTP, and UDP-glucose pyrophosphorylase

Before PFK regulatory step.

uridylyl transferase deficiency

galactokinase deficiency

epimerase deficiency (extremely rare)

Type III

Type I, because ATP will be depleted, and Galactose-1-phosphate will be accumulated

YES

The pyrophosphate resulting from UDP-glucose production can be hydrolyzed with inorganic pyrophosphatase to get the energy needed

LECTURE 5

It is involved in utilizing the energy of metabolite oxidation for otherwise endergonic reductive biosynthesis.
Used for biosynthesis of FA and cholesterol.

1. oxidative steps
2. Non-oxidative steps

seen in liver, adipose cells, RBCs: largely absent in muscle as they need energy quickly. This pathway is important in biosynthetic tissues

3 glucose-6-phosphate + 3NADP+ + 6H2O ---> 2 fructose-6-phosphate + glyceraldehyde-3-phosphate + 6 H+ + 3 CO2 + 6 NADPH

3 Ribulose-5-phosphate (Ru5P)

Ribose-5-phosphate (R5P) and 2 Xylulose-5-phosphate(Xu5P)

2 Fructose-6-phosphate and Glyceraldehyde-3-phosphate

The first one: the enzyme used, glucose-6-phosphate dehydrogenase, is highly specific for NADP+, and strongly inhibited by NADPH.

nucleotides

NADPH is needed to regenerate reduced glutathione from glutathione disulfide.