Handout 7- Catabolism: Glycolysis and variations on carbohydrate metabolism

Glycolytic pathways

Convert sugars to pyruvate. Embden-Meyerhof-Parnas (EMP) Pathway uses hexose sugars, and is the most common. Entner-Doudoroff (ED) Pathway uses sugar acids

Pentose Phosphate Pathway

Conversion of hexose sugars to pentose sugars and NADPH + H. Can also convert pentose sugars into glyceraldehyde-3P and dihydroxyacetone-P for entry into glycolysis

TCA cycle

Oxidation of acetyl-CoA into 2 CO2 and formation of GTP, 3 NADH+H, FADH2, and CoA-SH. Under anaerobic conditions only a subset of reactions associated with the TCA cycle occur but not the TCA cycle itself.

ATP synthesis via substrate-level phosphorylation

Have a reactant with a phosphate on it, then bring in ADP to transfer the phosphate onto, creating ATP and a dephosphorylated reactant.

Functions that the reactions of central carbon metabolism are involved in

Synthesis of precursory metabolites, synthesis of ATP by substrate-level phosphorylation, synthesis of NADPH+H for anabolic reactions, and synthesis of NADPH+H for oxidative phosphorylation.

Overall stoichiometry of EMP Pathway

Invest 1 glucose and 2 ATP to generate 2 net ATP, 2 NADH+H and 2 pyruvate

Glucokinase

(EMP and ED) Converts glucose to G6P, using ATP as it does so. This enzyme is only used during times of stress, otherwise PEP is used. This step is irreversible

Phosphoglucose isomerase (pgi)

(EMP) Converts G6P to F6P.

Phosphofructokinase-1 (PFK)

(EMP) Converts F6P to Fructose 1.6-bisphosphate, using ATP as it does so. This step is irreversible. This enzyme is allosterically regulated, making this the rate limiting step of glycolysis

Aldolase (fbaA/B)

(EMP) Converts Fructose 1.6-bisphosphate to G3P and dihydroxyacetone phosphate. fbaA is only expressed when grown on glucose. fbaB is only expressed during gluconeogenesis

Triose phosphate isomerase (tpiA)

(EMP) Converts g3p and dihydroxyacetone phosphate into 2 molecules of G3P

Glyceraldehyde 3-P dehydrogenase (gapA)

(EMP) Converts 2 molecules of g3p to 2 molecules of 1,3-bisphosphoglycerate, creating 2 molecules of NADH+H as it does so

Phosphoglycerate kinase (pgk)

(EMP) Converts 2 molecules of 1,3-bisphosphoglycerate to 2 molecules of 3-phosphoglycerate, making 2 molecules of ATP as it does so.

Phosphoglycerate mutase (pgm)

(EMP) Converts 2 molecules of 3-phosphoglycerate to 2 molecules of 2-phosphoglycerate

Enolase (eno)

(EMP) Converts 2 molecules of 2-phosphoglycerate to 2 molecules of phosphoenolpyruvate, creating 2 H2O as it does so

Pyruvate kinase (pykA/F)

(EMP) Converts 2 molecules off phosphoenolpyruvate to 2 molecules of pyruvate, creating 2 ATP as it does so. This step is irreversible.

Glycolysis enzymes unique to EMP

Glucokinase, phosphoglucose isomerase (pgi), phosphofructokinase (pfkA/B), aldolase (fbaA/B), triose phosphate isomerase (tpiA)

Glycolysis reactions common to both EMP and ED

Glyceraldehyde 3-P dehydrogenase (gapA), phosphoglycerate kinase (pgk), phosphoglycerate mutase (pgm), enolase (eno), pyruvate kinase (pykA/F)

Irreversible steps of glycolysis

Glucokinase, Phosphofructokinase (pfkA/B), and Pyruvate kinase (pykA/F) reactions

Enzymes as catalysts

Protein catalysts. Influence the kinetics, but not the thermodynamics of a reaction. Increase the reaction rate by lowering activation energy but do not alter the equilibrium.

Delta G being larger than 0

The reaction not favorable/spontaneous

Delta G is less than 0

The reaction is favorable/spontaneous

Delta G equals 0

The reaction is at equilibrium

Standard conditions

[1M] of all reactants and products, pH = 7, 25 C

Delta g equation

Delta G= Detla Go’ = RTlnQ

Q

The reaction quotient, a ration of the concentration of reactants to products

Delta G at equilibrium

Delta G= 0 = Delta Go’+RTlnKeq, where Keq is the equilibrium constant, which is the ratio of products to reactants at equilibrium

Km

The Menten constant, which is the concentration of substrate at which th enzyme is working at half capacity

Concentrations of metabolites and thermodynamics of glycolysis

The metabolites of early steps of glycolysis are at high concentrations, whereas metabolites of later stages of glycolysis are a low concentrations. This helps drive glycolysis forward

Enzymes for degradation pathways

Have Km » [S] so they don’t degrade important metabolites like amino acids unless they become too abundant

Enzymes for synthesis pathways

Have Km « [S] so they are working a full capacity to get the most out of each enzyme produced

Enzymes of central carbon metabolism

Have Km near the substrate concentration so that they maintain a reasonable substrate concentration and are able to work in both directions

Delta G of reversible enzymes

Very close to zero so that the direction of the reactions can be easily switched

Delta G of irreversible reactions

Very far from zero so that the reactions can only go one way.

Primary method for phosphorylating glucose in E. coli

Not glucokinase, but rather the phosphotransferase system (PTS)

Why is the first step of glycolysis reversible

Irreversible conversion of glucose into hexose phosphate by the first step traps metabolites in the cells and keeps intracellular glucose concentrations low to favor glucose uptake

Phosphotransferase system (PTS) in E. coli

Takes up a phosphate from PEP and transfers it through a chain of proteins until it is eventually transferred to glucose, creating G6P.

EIIA

The enzyme in the PTS system that performs inducer exclusion and carbon catabolite repression

Inducer Exclusion

The part of the PTS system that blocks import and utilization of other carbon sources when glucose is present. EIIA-Glc (that has already given its phosphate to glucose) serves as the allosteric regulator in this case to block other c-sources being utilized

Carbon Catabolite Repression

A part of the PTS system regulates whether genes for utilizing other c-sources are transcribed. EIIA-Glc-P building up (because no glucose is present to donate the P to) interferes with adenylate cyclase, causing cAMP to be produced. cAMP binds to cAMP receptor protein (crp) which is a transcription regulator, turning on the genes for utilizing other c-sources

FBP

Gluconeogenesis enzyme found in both e. coli and b. subtilis. Interconverts between f-1,6 bisP and F6P

gapA/B

B subtilis has 2 distinc g3p ddehydrogenase enzymes. gapA is used for glycolysis and converts g3p to 1,3 bisphosphorogylcerate. gapB is used for gluconeogenesis and converts 1,3 bisphosphoroglycertate to g3p

Pyruvate-water dikinase (PpsA)

Gluconeogenesis enzyme that is unique to e. coli, and interconverts pyruvate and PEP

PEP carboxykinase (pck)

Gluconeogenesis enzyme found in both e coli and b subtilis that converts oxaloacetate to PEP

PEP carboxylase (ppc)

Gluconeogenesis enzyme unique to e coli that converts PEP to oxaloacetate

Pyruvate carboxylase (Pyc)

Gluconeogenesis enzyme unique to b subtilis that converts pyruvate to oxaloacetate

Malate dehydrogenase (mae)

Gluconeogenesis enzyme found in both e. coli and b. subtilis that interconverts between malate and pyruvate

Glucose-6-phosphate dehydrogenase

(ED) Converts G6P to 6-phosphogluconolactone

Lactonohydrolase

(ED) Converts 6-phosphogluconolactone and water into 6-phosphogluconate

6-phosphogluconate dehydratase

(ED) Converts 6-phosphogluconate into water and KDPG

KDPG Aldolase

(ED) Splits KDPG into pyruvate and G3P, rest of EMP pathway is followed like normal

Similarities between ED and PPP

Share the same first three steps, catalyzed by glucokinase/PTS system, glucose-6-phosphate dehydrogenase and lactonohydrolase

Unique reactions to ED pathway

the reactions catalyzed by 6-phosphogluconate dehydratase and KDPG Aldolase to make G3P and pyruvate

Consumption/Production in EMP pathway

Overall +2 ATP and 2 NADH. 2 ATP consumed in first branch, then 4 ATP and 2 NADH produced in the second branch

Overall consumption/production in ED pathway

Overall 1 ATP, 1 NADH, and 1 NADPH created. 1 ATP consumed and 1 NADPH created in the ED branch, then 2 ATP and 1 NADH made in lower EMP

Archaeal EMP and ED pathways

Carry energy in forms other than ATP, because when you heat ATP to high heats the bonds break. Use cofactors like ferredoxin instead.

GAPOR and GAPN

The enzymes that archaea use in EMP/ED to replace Gap dehydrogenase and phosphoglycerate kinase. Ultimately convert G3P to 3PG in the semi-phosphorylating ED branch

Modified ED pathways in archaea

Can utilize either the semi-phosphorylating or non-phosphorylating ED branches.

Semi-phosphorylating ED branch in archaea

Brings in one phosphate, and uses the enzymes GAPN and GAPOR

Non-phosporylating ED branch in archaea

Never brings in a single phosphate, and uses the enzymes GAOR and GADH to convert from glyceraldehyde to Glycerate, which then gets converted to 2PG to run out the rest of ED normally

Input to the pentose phosphate pathway

Can be glucose oxidized to 6-phosphogluconate (like in ED pathway), or F6P and GAP from glycolysis and gluconeogenesis

Importance of transketolases and transaldolases

Allow the shuttling and recombining of compounds into and of other pathways

Oxidative phase of PPP

Involves the first 3 reactions, ultimately converting G6P to R5P

Non-oxidative phase of PPP

Involves the conversion of R5P to xylulose-5-P and ribose-5-P via RPI and enzymes, then transketolase and transaldolase reactions that ultimately result in the production of fructose-6-P and P-glyceraldehyde

6-P-gluconate dehydrogenase

(PPP) Converts 6-P-gluconate to Ribulose-5-phosphate, creating NADPH+H and CO2 as byproducts

Ribulose-5-phospshate epimerase

(PPP) Converts Ribulose-5-P to xylulose-5-P

Riibose-5-P isomerase

Converts ribulose-5-p to ribose-5-p

Transketolase (first use)

(PPP) Combines xylulose 5-p and ribose-5-p to create phosphoglyceraldehyde and sedoheptulose

Transaldolase

(PPP) Combines phosphoglyceraldehyde and sedoheptulose-7-P t create fructose-6-P and erythrose-4-p

Transketolase (second use)

(PPP) Combines erythrose-4-p and xylulose-5-p to create p-glyceraldehyde and fructose-6-p

Purpose of PPP

Can create precursory metabolites in the form of ribose-5-phosphate and erythrose-4-phosphate for aromatic amino acids. Can also generate NADPH+H to be used for biosynthesis reactions. How much of each of these pulled out of the pathway depends on cellular needs.

Excess glucose in the PPP

Can be pulled out and reenter EMP as F6P and G3P to still make some energy. Overall makes 5/3 ATP per glucose, which is in between what you would make in EMP or ED

Purpose of the TCA cycl

Oxidation of Acetyl-CoA to 2 CO2 and CoA, as well the creation or reducing equivalents in the form of NADH and Q for energy metabolism

Citrate Synthase

(TCA) Takes oxaloacetate, acetyl-CoA, and H2O, and creates citrate, CoA, and H+

Aconitase

(TCA) Converts citrase to H2O and cis-Acconitate, then adds H2O back in to create Isocitrate

Isocitrate dehydrogenase

(TCA) The first committed step of the TCA cycle, as it is allosterically regulated by protein phosphorylation (the rate limiting step). It converts isocitrate and NADP to NADPH, Co2, and a-ketoglutarate. Irreversible

a-ketoglutarate dehydrogenase

(TCA) Converts a-ketoglutarate, NAD+, and CoA into succinyl-CoA , NADH, and CO2. This rxn is irreversible

Succinyl-coa synthetase

(TCA) Converts Succinyl-CoA, ADP, and Pi to ATP, CoA, and Succinate

Succinate dehydrogenase

(TCA) Converts succinate and Q (or FAD in euks) to Fumarate and QH2 (or FADH2)

Fumarase

(TCA) Converts fumarate and h2o into malate

Malate dehydrogenase

(TCA) Converts malate and NAD into NADH+H and oxaloacetate

Overall TCA rxn

Acetyl-CoA + 3 NAD + Q + GDP + Pi to 2 CO2 + 3 NADH + 3H + QH2 + GTP + CoA

Committed steps in the TCA cycle

Pull of electrons and generate CO2. These include the reactions carried out by isocitrate dehydrogenase and a-ketoglutarate dehydrogenase

How does the TCA cycle start

With oxaloacetate and Acetyl-CoA. Enzyme Pyruvate dehydrogenase takes pyruvate and ultimately creates Acetyl-CoA. Oxaloacetate is generated by PEP Carboxylase in E. coli and Pyruvate carboxylase in b. subtilis. These are all anapleurotic reactions

Pyruvate Dehydrogenase Structure

A complex of 3 enzymes, called E1, E2, and E3

Reaction mechanism of Pyruvate Dehydrogenase

TPP cofactor in E1 accepts the acetyl group from pyruvate and releases CO2. E2 transfers the acetyl group onto CoA-SH, making and releasing Acetyl-CoA. E3 regenerates electrons and oxidizes the cofactor of E2 so the process can repeat