Gluconeogenesis, amino acids and lipids

Describe why the conversion of pyruvate to PEP is difficult

• The reverse is a SLP as the phosphate has a good LG

Describe the action of pyruvate carboxylase

• Conversion of pyruvate to oxaloacetate

• Furst step uses one molecule of ATP and Carbon dioxide

• Forming a carbon-carbon bond as we are adding a Co2 on the methyl terminus of the pyruvate

What does CO2 do in solution? Why is this bad

• IN water CO2 spontaneously forms carbonate

• For reaction need it in the electrophilic form, so need to have one of the oxygens as a good LG

Describe the mechanism of pyruvate carboxylase

• Carbonate is -vely charged so acts as a nucleophile and attacks the terminal phosphate of an ATP

• SN2 at the phosphorous, kicking out ADP

• Forms an anhydride

By adding the phosphate, the oxygen is now a good LG due to resonance

• Water can now acts as a base

• Deprotonates the acidic hydrogen

• E2 reaction to form CO2 which is electrophilic and phosphate

• In cells there is lots of water so the CO2 will reform the carbonic acid

• Double bond of biotin is electron rich as the oxygen donates electrons in

• Can attach the carbon dioxide

• Forms a carbamate - this is a reversible way of forming the CO2 until the carboxylase is ready to use it

• Pyruvate deprotonated in the alpha position

• Forms an enolate

• Enolate causes a conformational shift, brining the biotin into the correct position of the active site

• Can then decarboxylate the biotin

• Electrons can be kicked onto the nitrogen as they can resonate onto the other carbonyl

• Enolate form of the pyruvate can attack the carbon dioxide via nucleophilic attack

--> oxygen -ve charge can donate electrons onto the alkene

• Forms oxaloacetate

• Proton transfer allow biotin to be reformed

• Biotin reformed

Describe the role of biotin

• Acts as a holder of CO2 so we don’t need to keep dehydrating it (lots of ATP needed)

• Bacteria in the microbiome produce it, some people have a metabolic issue with it

• Has a similar carboxylic acid to lipoamide that allows linkage to a lysine, may have issues with the enzyme that links it

• Hydrogens on the nitrogen's are quite acidic as they are alpha to a carbonyl

• So they can be deprotonated and form an N=C and O-

Describe the conversion of oxaloacetate to malate out of the mitochondria and why it occurs

• Occurs by membrane crossing

• Transferring out of the mitochondria into the cytosol of the cell

• In the mitochondria, NADH reduces to malate

• Malate crosses membrane into the cytosol

• Malate is then oxidised to oxaloacetate

• Changes the ratio of NADH and NAD+ in the cytosol and the mitochondria

--> more NAD+ in the mitochondria needed

--> more NADH into the cytosol as reducing equivalents are needed for gluconeogenesis

Describe the reaction of PEP carboxykinase

• Conversion of oxaloacetate to PEP

• Uses a GTP as a source of the phosphate

• A carbon dioxide can be removed again - the one added can be removed

• CO2 is used as a catalyst with the carboxykinase and cabroxylase

Describe the mechanism of PEP carboxykinase

• Oxaloacetate can spontaneously decarboxylate

• Move e- in and they can then resonate up into the carbonyl

• Enzyme puts the GTP in the correct position, carbonyl O acts as a nucleophile to kick out the GDP

• We have formed PEP, CO2 and GDP

--> able to do as the carbonyl is in the correct relative position

 

Describe the reaction of fructose bisphosphatase

Fructose 1,6bisphosphate to fructose 6 phosphate

Describe the reaction of glucose 6 phosphatase

• Conversion of glucose6phosphate to glucose

• Cant to SLP as there is a bad LG

 

Describe the mechanism of glucose 6 phosphatase

• Phosphorylate the histidine, histidine acts as a nucleophile (resonates one N lone pair onto the other)

• Alcohol can be protonated as it leaves

• Phosphate is still covalently linked to the enzyme

• Hydrolyse the phosphate

• Histidine acting as an acid in the first acts as a base

• Deprotonates water which can attack phosphorous and regenerate histidine

• Formed inorganic phosphate and regenerated residues for next round

Why does gluconeogenesis occur?

• Helps stabilise blood glucose levels

• In ruminants, they are constantly doing gluconeogenesis due to their different stomachs

Describe how deaminase work?

• Have a nitrogen amino group

• Use deaminases to remove the amino group

• Use a pyridoxal cofactor, when this reacts with an amino acid it forms pyridoxamine

• Alanine can be converted to pyruvate, so have an alternate store, don’t need to glycolysis

 

Describe the mechanism of the pyridoxal enzyme complex

• Lysine amino group is deprotonated, attacks the carbonyl which is protonated

• Form a tetrahedral intermediate

• Enzyme presents a proton to protonate the alcohol and kick the water out, forming a C=N

• Covalent link holds the pyridoxal in the right place

Describe how a deaminase works

• Transamination rection can occur

• One imine converted to another

• Kickout the lysine and add the amino group of the alanine

• Alanine NH2 deprotonated by the enzyme so can attack the electron deficient carbon

 

• Tetrahedral intermediate forms and imine is formed, kick out the lysine and reprotonates

 

• Not overly reactive due to lack of charges

• Protonate the nitrogen on the pyridine ring

• Push electrons down to neutralise the +ve charge

• Deprotonation a to the nitrogen of the amino acid

• Kick electrons through the ring to neutralise negative charge

 

 

• Have now formed another imine

• Can reprotonates at the top and deprotonate at the bottom

• Reformation of aromaticity is driving the step

• Imine hydrolysis

• Deprotonate water and attack the C=N, breaks this and can accept a proton

• Tetrahedral intermediate

• Protonate nitrogen to make a good LG and deprotonate oxygen

• Forms a carbonyl

• Removed a nitrogen and oxidised it

• Pyridoxal has gained a nitrogen and been reduced

Describe how pyridoxal can be regenerated

• Need to generate pyridoxal

• One mechanism mechanism uses NAD+ to oxidise

• Nitrogen forms ammonia- ammonia is toxic, doesn’t do this loads as toxic

• The urea cycle

• Carbonate and ammonia react with a phosphate

• Form lots of intermediates, these eventually form urea